Therapeutic drug for malignant tumors

ABSTRACT

According to the present disclosure there are provided compositions and methods for treating malignant tumors, including an anti-LSR (lipolysis stimulated lipoprotein receptor) antibody that comprises the presently disclosed antibody heavy and light chain complementarity determining region (CDR) sequences, or an antigen-binding fragment thereof, or a functional equivalent thereof. Further provided for treating an LSR-positive malignancy is an LSR antagonist or an LSR inhibitor such as a nucleic acid. Therapeutic administration of the anti-LSR antibody to a subject having an LSR-positive malignant tumor is also described.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is 690188_402D1_SEQUENCE_LISTING.txt. The text fileis 32.9 KB, was created on Oct. 2, 2018, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to a therapeutic drug, diagnostic drug orthe like for malignant tumor.

BACKGROUND ART

LSR (lipolysis stimulated lipoprotein receptor) is known as a moleculeassociated with metabolism of low-density lipoprotein (LDL). Several LSRrelated research results have been reported. For example, Non PatentLiterature 1 describes that LSR expression is reduced in a liver ofobese and type 2 diabetes mouse models. Further, Non Patent Literature 2describes that LSRs are expressed in bladder cancer. Non PatentLiterature 3 describes that LSRs are expressed in colon cancer cells.Non Patent Literature 4 describes that LSRs are expressed in breastcancer cells. Patent Literature 1 describes that LSRs are expressed inovarian cancer cells or the like.

CITATION LIST Patent Literature

-   [PTL 1] WO 2012/140627

Non Patent Literature

-   [NPL 1] “Liver-specific loss of lipolysis-stimulated lipoprotein    receptor triggers systemic hyperlipidemia in mice.” Narvekar et al.,    Diabetes. 2009 May; 58(5): 1040-9-   [NPL 2] “Increased cell motility and invasion upon knockdown of    lipolysis stimulated lipoprotein receptor (LSR) in SW780 bladder    cancer cells.” Herbsleb et al., BMC Med Genomics. 2008 Jul. 22;    1:31.-   [NPL 3] “Prognostic value of LISCH7 mRNA in plasma and tumor of    colon cancer patients.” Garcia et al., Clin Cancer Res. 2007 Nov. 1;    13(21): 6351-8.-   [NPL 4] “Functional heterogeneity within the CD44 high human breast    cancer stem cell-like compartment reveals a gene signature    predictive of distant metastasis.” Leth-Larsen et al., Mol Med. 2012    Sep. 25; 18: 1109-21.

SUMMARY OF INVENTION Solution to Problem

The disease mechanism of malignant tumor is complex, with many partsunclear. Thus, many medical needs remain unfulfilled in the field. Theinventors have discovered that growth of malignant tumor cells issuppressed when the malignant tumor cells are contacted with an anti-LSRantibody as a number of researches are conducted on malignant tumor.Furthermore, when an LSR siRNA was transfected into malignant tumorcells, growth of malignant tumor cells was also suppressed in this case.In addition, when an anti-LSR antibody was actually administered to amalignant tumor model mouse, a notable decrease in tumor volume wasobserved. In view of the above, it was elucidated that an LSRsuppressant such as an anti-LSR antibody is effective in treatingmalignant tumor. In this regard, the present invention provides a noveltherapeutic agent for malignant tumor targeting LSRs and the like.

Further, the inventors elucidated, as described in the Examplesdisclosed below, the presence of many LSR negative patients while thereare LSR positive patients among malignant tumor patients. In view of theabove, it was elucidated that diagnosis of the presence or absence ofLSR positive state in a malignant tumor patient prior to therapy isimportant in the treatment of malignant tumor targeting LSRs.

The Examples of the above-described Patent Literature 1 suggest that anLSR mRNA was detected in a few types of cancer. The claims have anactual recitation of an anti-LSR antibody inducing apoptosis of cancercells. However, Patent Literature 1 does not have any pharmacologicaldata from an actual successful cancer therapy. In addition, PatentLiterature 1 does not describe that the presence or absence of an LSRpositive condition in a malignant tumor patient is diagnosed prior totherapy. For this reason, an anti-LSR antibody could not be consideredeffective for treating malignant tumor only from the results of PatentLiterature 1.

In one aspect, the present invention provides a therapeutic orprophylactic drug for malignant tumor, comprising a suppressant of anLSR (lipolysis stimulated lipoprotein receptor).

In one embodiment, the suppressant in the present invention can comprisean anti-LSR (lipolysis stimulated lipoprotein receptor) antibody, anantigen binding fragment or a functional equivalent thereof, or anucleic acid.

In another embodiment, the suppressant in the present invention cancomprise an anti-LSR (lipolysis stimulated lipoprotein receptor)antibody or an antigen binding fragment or a functional equivalentthereof.

In still another embodiment, the suppressant in the present inventioncan be an RNAi molecule directed to an LSR or a polynucleotide encodingthe RNAi molecule.

In still another embodiment, the malignant tumor in the presentinvention can be LSR positive malignant tumor.

In still another embodiment, the present invention can be foradministration to a patient determined to have an episode of LSRpositive malignant tumor.

In still another embodiment, the present invention can be foradministration to a patient among malignant tumor patients whosemalignant tumor has been determined to be LSR positive malignant tumor.

In still another embodiment, the anti-LSR antibody in the presentinvention can be an anti-LSR antibody that specifically binds to anepitope of an LSR. More specifically, the antibody may have positions116-134 and/or 216-230 of SEQ ID NO: 7 as the epitope.

In still another embodiment, the anti-LSR antibody in the presentinvention can be an antibody having an ability to inhibit exacerbationdue to a VLDL.

In still another embodiment, the anti-LSR antibody in the presentinvention may be one or more antibodies selected from the groupconsisting of: (a) an antibody with heavy chain CDRs 1, 2, and 3 andlight chain CDRs 1, 2, and 3 comprising amino acid sequences set forthin positions 31-35, 50-66, 99-104, 153-165, 182-188 and 221-230 of SEQID NO: 1, respectively; (b) an antibody with heavy chain CDRs 1, 2, and3 and light chain CDRs 1, 2, and 3 comprising amino acid sequences setforth in positions 31-35, 50-66, 99-103, 152-165, 182-188 and 221-230 ofSEQ ID NO: 2, respectively; (c) an antibody with heavy chain CDRs 1, 2,and 3 and light chain CDRs 1, 2, and 3 comprising amino acid sequencesset forth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and221-229 of SEQ ID NO: 3, respectively; (d) an antibody with heavy chainCDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising amino acidsequences set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188and 221-229 of SEQ ID NO: 4, respectively; (e) an antibody with heavychain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising aminoacid sequences set forth in positions 31-35, 50-66, 99-104, 153-165,182-188 and 221-229 of SEQ ID NO: 5, respectively; and (f) an antibodywith heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3comprising amino acid sequences set forth in positions 31-35, 50-66,99-104, 153-165, 182-188 and 221-229 of SEQ ID NO: 6, respectively, or amutant of the antibody, which is free of a mutation in the CDRs butcomprises one or several substitutions, additions, or deletions in aframework of the antibody in the mutant.

In still another embodiment, the anti-LSR antibody in the presentinvention can be a monoclonal antibody.

In still another embodiment, an antibody class of the anti-LSR antibodyin the present invention may be IgG.

In still another embodiment, the anti-LSR antibody in the presentinvention may be an antigen binding fragment.

In another aspect, the present invention provides an agent forsuppressing cell division of a malignant tumor cell, comprising ananti-LSR antibody.

In another aspect, the present invention provides a companion diagnosticdrug for malignant tumor therapy targeting an LSR, comprising an LSRdetection agent.

In one embodiment, the LSR detection agent in the present invention cancomprise an anti-LSR antibody. In another aspect, the present inventionprovides a companion diagnostic method for malignant tumor therapytargeting an LSR, comprising inspecting whether a malignant tumor sampleof a malignant tumor patient is LSR positive. In another aspect, thepresent invention provides an antibody or antibodies selected from thegroup consisting of: (a) an antibody with heavy chain CDRs 1, 2, and 3and light chain CDRs 1, 2, and 3 comprising amino acid sequences setforth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and 221-230 ofSEQ ID NO: 1, respectively; (b) an antibody with heavy chain CDRs 1, 2,and 3 and light chain CDRs 1, 2, and 3 comprising amino acid sequencesset forth in positions 31-35, 50-66, 99-103, 152-165, 182-188 and221-230 of SEQ ID NO: 2, respectively; (c) an antibody with heavy chainCDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising amino acidsequences set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188and 221-229 of SEQ ID NO: 3, respectively; (d) an antibody with heavychain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising aminoacid sequences set forth in positions 31-35, 50-66, 99-104, 153-165,182-188 and 221-229 of SEQ ID NO: 4, respectively; (e) an antibody withheavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprisingamino acid sequences set forth in positions 31-35, 50-66, 99-104,153-165, 182-188 and 221-229 of SEQ ID NO: 5, respectively; and (f) anantibody with heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2,and 3 comprising amino acid sequences set forth in positions 31-35,50-66, 99-104, 153-165, 182-188 and 221-229 of SEQ ID NO: 6,respectively, or a mutant of the antibody, which is free of a mutationin the CDRs but comprises one or several substitutions, additions, ordeletions in a framework of the antibody in the mutant. These antibodiesmay be an antibody selected from a monoclonal antibody, polyclonalantibody, chimeric antibody, humanized antibody, human antibody,multifunctional antibody, bispecific or oligospecific antibody, singlechain antibody, scFV, diabody, sc(Fv)₂ (single chain (Fv)₂), andscFv-Fc.

In another aspect, the present invention provides a composition forpreventing or treating malignant tumor, comprising an LSR binding agent.In one embodiment, the malignant tumor in the present invention can beLSR positive malignant tumor.

In another embodiment, the present invention can further comprise acell-killing agent.

In another embodiment, the LSR binding agent in the present inventionmay be an antibody, a fragment or a functional equivalent thereof, or anucleic acid. In a specific embodiment, the LSR binding agent in thepresent invention may be an antibody or a fragment or a functionalequivalent thereof, further bound to a cell killing agent. In a specificembodiment, the malignant tumor in the present invention may compriseovarian cancer. The ovarian cancer in the present invention may berecurrent ovarian cancer. Alternatively, the malignant tumor may bemetastasized ovarian cancer. The malignant tumor can comprise ovariancancer, pancreatic cancer, lung cancer, gastric cancer, or colon cancer.Alternatively, the malignant tumor may be early-stage ovarian cancer. Inanother embodiment, ovarian cancer can be ovarian serous adenocarcinomaor ovarian clear cell adenocarcinoma. In a specific embodiment, the LSRbinding agent in the present invention may be characterized by having anantibody or a fragment or a functional equivalent thereof, the antibodybeing one or more antibodies selected from the group consisting of: (a)an antibody with heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2,and 3 comprising amino acid sequences set forth in positions 31-35,50-66, 99-104, 153-165, 182-188 and 221-230 of SEQ ID NO: 1,respectively; (b) an antibody with heavy chain CDRs 1, 2, and 3 andlight chain CDRs 1, 2, and 3 comprising amino acid sequences set forthin positions 31-35, 50-66, 99-103, 152-165, 182-188 and 221-230 of SEQID NO: 2, respectively; (c) an antibody with heavy chain CDRs 1, 2, and3 and light chain CDRs 1, 2, and 3 comprising amino acid sequences setforth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and 221-229 ofSEQ ID NO: 3, respectively; (d) an antibody with heavy chain CDRs 1, 2,and 3 and light chain CDRs 1, 2, and 3 comprising amino acid sequencesset forth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and221-229 of SEQ ID NO: 4, respectively; (e) an antibody with heavy chainCDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising amino acidsequences set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188and 221-229 of SEQ ID NO: 5, respectively; and (f) an antibody withheavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprisingamino acid sequences set forth in positions 31-35, 50-66, 99-104,153-165, 182-188 and 221-229 of SEQ ID NO: 6, respectively, or a mutantof the antibody, which is free of a mutation in the CDRs but comprisesone or several substitutions, additions, or deletions in a framework ofthe antibody in the mutant.

In still another embodiment, the anti-LSR antibody is an antibodyselected from a monoclonal antibody, polyclonal antibody, chimericantibody, humanized antibody, human antibody, multifunctional antibody,bispecific or oligospecific antibody, single chain antibody, scFV,diabody, sc(Fv)₂ (single chain (Fv)₂), and scFv-Fc.

That is, according to another aspect of the present invention, atherapeutic drug for malignant tumor comprising an anti-LSR antibody isprovided.

Further, according to another aspect of the present invention, atherapeutic drug for malignant tumor, comprising an LSR antagonist isprovided.

Further, according to another aspect of the present invention, an agentfor suppressing cell division of a malignant tumor cell, comprising ananti-LSR antibody is provided.

Further, according to another aspect of the present invention, acompanion diagnostic drug for malignant tumor therapy targeting an LSR,comprising an anti-LSR antibody is provided. One embodiment ischaracterized in that the malignant tumor is determined to be LSRpositive by the companion diagnostic method of present invention, andthe LSR binding agent is administered thereafter.

Further, according to another aspect of the present invention, acompanion diagnostic method for malignant tumor therapy targeting anLSR, comprising inspecting whether a malignant tumor sample of amalignant tumor patient is LSR positive, is provided.

In a specific embodiment, the malignant tumor may be LSR positivemalignant tumor. Further, in one embodiment of the present invention,the above-described therapeutic drug may be a therapeutic drug foradministration to a patient determined to have an episode of LSRpositive malignant tumor. Further, in one embodiment of the presentinvention, the above-described therapeutic drug may be a therapeuticdrug for administration to a patient among tumor patients whosemalignant tumor has been determined to be LSR positive malignant tumor.Further, in one embodiment of the present invention, the anti-LSRantibody may be an anti-LSR antibody that specifically binds to anepitope of an LSR. Further, in one embodiment of the present invention,the anti-LSR antibody may be one or more antibodies selected from thegroup consisting of: (a) an antibody with heavy chain CDRs 1, 2, and 3and light chain CDRs 1, 2, and 3 comprising amino acid sequences setforth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and 221-230 ofSEQ ID NO: 1, respectively; (b) an antibody with heavy chain CDRs 1, 2,and 3 and light chain CDRs 1, 2, and 3 comprising amino acid sequencesset forth in positions 31-35, 50-66, 99-103, 152-165, 182-188 and221-230 of SEQ ID NO: 2, respectively; (c) an antibody with heavy chainCDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising amino acidsequences set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188and 221-229 of SEQ ID NO: 3, respectively; (d) an antibody with heavychain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising aminoacid sequences set forth in positions 31-35, 50-66, 99-104, 153-165,182-188 and 221-229 of SEQ ID NO: 4, respectively; (e) an antibody withheavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprisingamino acid sequences set forth in positions 31-35, 50-66, 99-104,153-165, 182-188 and 221-229 of SEQ ID NO: 5, respectively; and (f) anantibody with heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2,and 3 comprising amino acid sequences set forth in positions 31-35,50-66, 99-104, 153-165, 182-188 and 221-229 of SEQ ID NO: 6,respectively, or a mutant of the antibody, which is free of a mutationin the CDRs but comprises one or several substitutions, additions, ordeletions in a framework of the antibody in the mutant. These antibodiesmay be an antibody selected from a monoclonal antibody, polyclonalantibody, chimeric antibody, humanized antibody, human antibody,multifunctional antibody, bispecific or oligospecific antibody, singlechain antibody, scFV, diabody, sc(Fv)₂ (single chain (Fv)₂), andscFv-Fc. The antibodies used are not limited, but an antibody havingpositions 116-134 and/or 216-230 of SEQ ID NO: 7 as an epitope can beadvantageously used. This is because an advantageous effect as well assafety and stability thereof are demonstrated herein.

Further, in a specific embodiment of the present invention, theabove-described anti-LSR antibody may be a monoclonal antibody. Further,in one embodiment of the present invention, an antibody class of theabove-described anti-LSR antibody may be IgG. Further, in one embodimentof the present invention, the above-described anti-LSR antibody may bethe antigen binding fragment. Further, in one embodiment of the presentinvention, the above-described LSR antagonist may be an RNAi moleculedirected to an LSR or a polynucleotide encoding the RNAi molecule.

In another aspect of the present invention, the present inventionprovides a poor prognosis marker for malignant tumor therapy, comprisingan LSR (lipolysis stimulated lipoprotein receptor) binding agent. It isunderstood that a binding agent in any form of the present inventionexplained herein can be used as the binding agent used in this aspect.For example, the binding agent may be an antibody, a fragment or afunctional equivalent thereof, or a nucleic acid, which may be labeled.

In another aspect of the present invention, the present inventionprovides a method of using an expression level of an LSR (lipolysisstimulated lipoprotein receptor) as an indicator for poor prognosis ofmalignant tumor therapy. It is understood that a binding agent in anyform of the present invention explained herein can be used as thebinding agent used in this aspect. For example, the binding agent may bean antibody, a fragment or a functional equivalent thereof, or a nucleicacid, which may be labeled.

In another aspect of the present invention, the present inventionprovides a diagnostic agent for poor prognosis of malignant tumortherapy, comprising an LSR (lipolysis stimulated lipoprotein receptor)binding agent. It is understood that a binding agent in any form of thepresent invention explained herein can be used as the binding agent usedin this aspect. For example, the binding agent may be an antibody, afragment or a functional equivalent thereof, or a nucleic acid, whichmay be labeled.

In still another aspect, the present invention provides a therapeuticmethod, prophylactic method, use and the like using a pharmaceuticalcomposition, therapeutic agent or prophylactic agent of the presentinvention.

It is understood that one or more of the aforementioned features can befurther combined for use.

Those skilled in the art who have read and understood the followingDetailed Description as needed would recognize further embodiments andadvantages of the present invention.

Advantageous Effects of Invention

According to the present invention, a novel therapeutic drug, diagnosticdrug or the like for malignant tumor is obtained.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a diagram showing results of RT-PCR performed on nucleic acidsobtained from ovarian serous adenocarcinoma cell strains. One on theleft show normal cells (HOSE2 on the left side). Three on the right showcancer cells (from the left: OVCAR3, OVSAHO, and JHOS4). The top rowshows LSRs and the bottom row shows the background β actin.

FIG. 2 is a diagram showing results of RT-PCR performed on nucleic acidsobtained from ovarian clear cell adenocarcinoma cell strains. One on theleft show normal cells (HOSE2C on the left side). Four on the right showcancer cells (from the left: OVTOKO, OVMANA, OVISE, and RMG-1). The toprow shows LSRs and the bottom row shows the background β actin.

FIG. 3 is a diagram showing results of RT-PCR performed on nucleic acidsobtained from endometrial cancer cell strains. The left end shows normalcells (E6/E7/TERT), and the others are cancer cells (from the left:HEC1, HEC1A, HEC6, HEC88nu, HEC108, HEC116, HEC251, and SNG-M). The toprow shows LSRs and the bottom row shows the background β actin.

FIG. 4 is a diagram showing results of Western blot performed onproteins obtained from ovarian serous adenocarcinoma cell strains. Oneon the left show normal cells (HOSE2C on the left side). Three on theright show cancer cells (from the left: OVCAR3, OVSAHO, and JHOS4).

FIG. 5 is a diagram showing results of Western blot performed onproteins obtained from ovarian clear cell adenocarcinoma cell strains.One on the left show normal cells (HOSE2C on the left side). Four on theright show cancer cells (from the left: OVTOKO, OVMANA, OVISE, andRMG-1).

FIG. 6 is a diagram showing results of Western blot performed onproteins obtained from endometrial cancer cell strains. The top left endshows normal cells (E6/E7/TERT), and others are cancer cells. The toprow shows, from the left, HEC1, HEC1A, HEC6, and HEC88nu. The bottom rowshows from the left, HEC108, HEC116, HEC251, and SNG-M. All of themrepresent LSRs.

FIG. 7 is a diagram showing results of Western blot performed onproteins obtained from tissue on which surgery has been performed forovarian serous adenocarcinoma and tissue on which surgery has beenperformed for ovarian clear cell adenocarcinoma. The Figure shows, fromthe left, two samples from normal (healthy individual's) ovaries (No. 1and No. 2=represented by (1) and (2)), two samples from clear celladenocarcinoma patients (No. 1 and No. 2=represented by (3) and (4)),and two samples from serous adenocarcinoma patients (No. 1 and No.2=represented by (5) and (6)).

FIG. 8 is a diagram showing results of Western Blot performed onproteins obtained from tissue on which surgery has been performed forendometrial cancer. The Figure shows, from the left, two samples fromnormal (healthy individual's) ovaries (No. 1 and No. 2=represented by(1) and (2)), and two samples from endometrial cancer patients (No. 1and No. 2=represented by (3) and (4)).

FIG. 9 is a diagram showing the amino acid sequence of the anti-LSRantibody described in the Examples.

FIG. 10 is a diagram showing results of assessing reactivity of #9-7antibody to, from the left, OVSAHO, JHSO4, RMG-I, and OVISE. Thevertical axis indicates intensity and the horizontal axis indicates cellfrequency.

FIG. 11 is a diagram showing results of assessing reactivity of #16-6antibody to, from the left, OVSAHO, JHSO4, RMG-I, and OVISE. Thevertical axis indicates intensity and the horizontal axis indicates cellfrequency.

FIG. 12 is a diagram showing results of assessing reactivity of #26-2antibody to, from the left, OVSAHO, JHSO4, RMG-I, and OVISE. Thevertical axis indicates intensity and the horizontal axis indicates cellfrequency.

FIG. 13 is a diagram showing results of assessing reactivity of #27-6antibody to, from the left, OVSAHO, JHSO4, RMG-I, and OVISE. Thevertical axis indicates intensity and the horizontal axis indicates cellfrequency.

FIG. 14 is a diagram showing results of assessing reactivity of #1-25antibody to, from the left, OVSAHO, JHSO4, RMG-I, and OVISE. Thevertical axis indicates intensity and the horizontal axis indicates cellfrequency.

FIG. 15 is a diagram showing a result of analyzing ovarian serousadenocarcinoma tissue for expression of LSRs by an immunohistochemicalstaining method using monoclonal the antibody #1-25. The top row showsserous G2 of ovary, serous G3 of tube, serous G3 of ovary and serous G3of ovary. All pictures in the bottom row show a clear cell of ovary.

FIG. 16 is a diagram showing a result of analyzing ovarian serousadenocarcinoma tissue for expression of LSRs by an immunohistochemicalstaining method using monoclonal antibodies #1-25. The top row showsstage IIIc of serous G2 of ovary, stage Ic of serous G3 of tube, stageIIb of serous G3 of ovary, and stage IIIb of serous G3 of ovary. Thebottom row shows, from the left, stage IIIc of clear cell of ovary,stage IIc of clear cell of ovary, and stage IV of clear cell of ovary.

FIG. 17 is a diagram showing a result of analyzing endometrial cancertissue for expression of LSRs by an immunohistochemical staining methodusing monoclonal antibodies #9-7 (4.5 μg/ml). The left side isendometrial cancer #1 applied with Toyobo Can get signal immunostainsolution A as the primary antibody diluent, and the right is endometrialcancer #1 applied with Toyobo Can get signal immunostain solution B asthe primary antibody diluent.

FIG. 18 is a diagram showing results of assessing #9-7 antibody for theeffect of suppressing growth of RMG-I. The vertical axis indicatesrelative growth compared with no treatment. The horizontal axis is thedosage of IgG.

FIG. 19 is a diagram showing results of assessing #1-25 antibody for theeffect of suppressing growth of RMG-I. The vertical axis indicatesrelative growth compared with no treatment. The horizontal axis is thedosage of IgG.

FIG. 20 is a diagram showing results of assessing #1-25 antibody (theleft three; from the left, 1 μg/ml, 10 μg/ml, and 100 μg/ml,respectively) and #26-2 antibody (middle three; from the left, 1 μg/ml,10 μg/ml, and 100 μg/ml, respectively) for the effect of suppressinggrowth of A2780. The control IgG is shown on the right (right three;from the left, 1 μg/ml, 10 fag/ml, and 100 μg/ml, respectively). Eachantibody has an effect, but #1-25 exhibited a stronger effect than#26-2.

FIG. 21 is a diagram showing results of assessing the LSR siRNAdescribed in the Examples for the effect of suppressing growth ofOVSAHO. From the left, day 1, day 2, day 3, day 4, and day 5 are shown.4 bars for each day indicate, from the left, mean amount with notreatment, mean value of control, mean value of LSR siRNA 1, and meanvalue of LSR siRNA2.

FIG. 22 is a diagram of a result showing that lipid (cholesterol)incorporation is elevated in the cells stably expressing LSRs describedin the Examples. The left shows the effects on total cholesterol, themiddle graph shows the effects on triglyceride, and the right shows theeffects on phospholipid. The vertical axis indicates each incorporation(mg/ml). The horizontal axis indicates each of EMP1 low density, EMP1high density, L45 low density, and L45 high density. EMP indicates emptyvector introduced cells and L indicates cells forced to express LSRs.

FIG. 23 is a diagram of a result showing that lipid (cholesterol)incorporation is elevated in high density culture in the cells stablyexpressing LSRs described in the Examples. The vertical axis indicatestotal cholesterol incorporation (mg/ml). EMP1 indicates empty vectorintroduced cells and L45 indicates cells forced to express LSRs.

FIG. 24 is a diagram of a result showing that LSR expression describedin the Examples elevates VLDL metabolism, but elevation in metabolismdue to VLDL is inhibited by LSR antibody administration. Significantinhibition of elevation in metabolism due to VLDL is exhibited for #9-7.Slight inhibition is also observed for the #1-25 antibody. In eachgraph, the vertical axis is OCR (pMoles/min) % and the horizontal axisindicates the elapsed time (minutes). Squares indicate PBS (backgroundcontrol), triangles indicate the control IgG, and rhombuses indicateanti-LSR antibodies. The top panel is for empty vector (E1) and thebottom is for cells forced to express LSRs (L45).

FIG. 25 is a diagram showing the condition when the anti-LSR antibodydescribed in the Examples was administered to a malignant tumor modelmouse.

FIG. 26 is a diagram showing results of assessing the anti-LSR antibodydescribed in the Examples for the antitumor effect after administrationof #9-7 or #1-25 to a malignant tumor model mouse. The vertical axisindicates tumor volume (mm³). The horizontal axis indicates the numberof elapsed days. Squares indicate the control IgG, triangles indicateanti-LSR antibody (#9-7), and rhombuses indicate anti-LSR antibody(#1-25).

FIG. 27 is a diagram showing results of assessing the anti-LSR antibodydescribed in the Examples for the antitumor effect after administrationof #9-7 or #1-25 to a malignant tumor model mouse. The vertical axisindicates tumor weight (mg). From the left, control IgG administeredgroup (n=8), anti-LSR antibody (#9-7) administered group (n=6), andanti-LSR antibody (#1-25) administered group (n=6) are shown.

FIG. 28 is a diagram showing results of assessing the anti-LSR antibodydescribed in the Examples for the antitumor effect after administrationof #9-7 or #1-25 to a malignant tumor model mouse. From the left,control IgG administered group (n=8), anti-LSR antibody (#9-7)administered group (n=6), and anti-LSR antibody (#1-25) administeredgroup (n=6) are shown.

FIG. 29 shows that recurrent ovarian cancer does not have an effectivetherapeutic method. Conventionally, there was no effective therapeuticmethod for recurrent ovarian cancer. The epidemiological characteristicof ovarian cancer is that ovarian cancer readily infiltrate into thesurrounding by the lymph node and peritoneal metastasis or the like andadvances quickly. For instance, 40% or more of ovarian cancer inJapanese patients is considered serous, 24% clear cells, 17%endometrioid, and 13% mucinous adenocarcinoma. As a 1st line of defense,cisplatin or taxol is used, and Avastin is used for recurrent ovariancancer. However, it was considered that improvement in survival rate wasnot observed. Since a therapeutic method during the progression stage orrecurrence is non-existent, ovarian cancer was considered as tumor withpoor prognosis. Thus, development of a novel therapeutic method isconsidered imperative. The Table on the left shows antibody medicamentsapproved as a cancer therapeutic drug (Carter P J Nat. Rev. Immunol.006, May 6 (5) 343-357, Review). The graph on the right side of FIG. 29shows the 5 year survival rate (31% in Stage IV) (Japanese Society ofObstetrics and Gynecology, Fujinka Shuyo Iinkai Hokoku [Gynecology tumorcommittee report], 2012, Vol. 64, No. 6).

FIG. 30 is an immunostaining diagram showing that LSRs are expressed inovarian cancer tissue. The left shows ovarian serous adenocarcinoma andthe right shows ovarian clear cell adenocarcinoma. The bottom panelshows Western blot of each cell. The left three columns show normalovary, columns 4-5 from the left show clear cell adenocarcinoma, andcolumn 6 from the left to the right end show serous adenocarcinoma. LSRindicates the band of LSRs, and GAPDH indicates the control.

FIG. 31 is a diagram showing that LSRs are also expressed in ovariancancer metastasized sites. The left column shows lymph node metastasisand the right column shows greater omentum metastasis. The top panelshows 100 times magnification and the bottom panel shows 400 timesmagnification.

FIG. 32 is a diagram showing that LSRs are also expressed in ovariancancer metastasized sites. The left column shows lymph node metastasisand the right column shows greater omentum metastasis. The top panelshows 100 times magnification and the bottom panel shows 400 timesmagnification.

FIG. 33 is a diagram showing that LSRs are expressed in ovary cancerfrom an early stage. The top left shows hematoxylin and eosin stain (HE)staining. Top middle shows #1-25A, top right shows #1-45A, bottom leftshows #9-7B, bottom middle shows #1-25B, and bottom right shows #1-45B.The cells shown are ovarian clear cells in Stage Ic/IIc.

FIG. 34 shows that LSRs are also specifically expressed in gastriccancer. The results of examination by immunostaining are shown, whichare results of immunostaining. The top row shows, from the left column,40 and 400 times magnification of ovarian clear cell cancer and 40 and400 times magnification of MK2 cells. The bottom row shows pictures of40 and 400 times magnificent of MK1 and 40 and 400 times magnificationof MK3.

FIG. 35 is a diagram showing that LSRs are strongly expressed in gastriccancer (signet ring cell cancer). The top left panel shows a picturemagnified 5 times, top right shows a picture magnified 10 times, bottomleft shows a picture magnified 20 times, and bottom right shows apicture magnified 40 times.

FIG. 36 is a diagram showing analysis of LSR expression by IHC using anormal frozen tissue array. The top row shows, from the left, adrenalgland, bone marrow, breast, brain (cerebellum), and brain (cerebralcortex). The middle row shows, from the left, brain (pituitary gland),colon, endothelium (aorta), endothelium (aorta, thoracic), andendothelium (artery). The bottom row shows, from the left, esophagus,fallopian tube, heart (right ventricle), kidney, and liver.

FIG. 37A is a diagram showing analysis of LSR expression by IHC using anormal frozen tissue array. The top row shows, from the left, lung,lymph node, ovary, pancreas, and placenta. The second row from the topshows, from the left, prostate, skin, spinal cord, spleen, and striatedmuscle. The second row from the bottom shows, from the left, stomach,testis, thymus, thyroid, and ureter. The bottom row shows, from theleft, endometrium and cervix.

FIG. 37B shows results of calculating the dissociation constant (K_(D))of anti-LSR antibodies by FACS. RMG-I cells were stained with antibodiesof various concentrations and analyzed by FACS. As shown, #9-7 hadK_(D)=2.52 nM, #1-25 had K_(D)=2.03 nM, #16-6 had K_(D)=2.33 nM, #26-2had K_(D)=4.04 nM, #27-6 had K_(D)=4.29 nM, and #1-43 had K_(D)=24.62nM.

FIG. 38 shows results of investigating prognosis of ovarian serousadenocarcinoma patients or ovarian clear cell adenocarcinoma based onwhether the LSR expression is high or low. 21 cases of patients withstrong expression of LSRs and 12 cases of patients with weak expressionwere studied for ovarian serous adenocarcinoma, and 27 cases of patientswith strong expression of LSRs and 24 cases of patients with weakexpression were studied for ovarian clear cell adenocarcinoma. It can beseen that ovarian serous adenocarcinoma with high level of expressionhas poorer prognosis compared to the group with low level of expression.

FIG. 39 shows a comparison of the epitope region of hLSR antibody of theantibody of the present invention with the amino acid sequence ofhLSR(SEQ ID NO: 21) and mLSR(SEQ ID NO: 22).

FIG. 40 shows that an anti-hLSR antibody cross-reacts with mLSR. Theoriginal diagram is shown in red and blue, where red indicates mIgG2a.Blue indicates the staining pattern in clones of various anti-LSRantibodies. Blue is marked with an arrow in this diagram. The reactionof various antibodies to COS7 cells subjected to transgenesis withpCMV5-mLSR-myc/DDK such that mSR is transiently expressed was confirmedby FACS. The top row shows mixture of COS7 and mLSR and the bottom rowshows only COS7 cells. Various antibodies are shown, which are from theleft, #9-7, #16-6, #26-2, #27-2, #1-25, and #1-43.

FIG. 41 shows that anti-LSR induces cell cycle arrest in RMG-I cells inthe G0/G1 phase. The graph shows the percentage of cells in the G0/G1phase, S phase and G2/M phase. For each phase, results for no treatment,treatment with the control IgG, and treatment with antibody #1-25 areshown from the left. The experiment was carried out with a 6 well plateat 15000 cells/well under the conditions of RPMI 1640 medium+1% FBS+1%penicillin-streptomycin (100 μg/ml antibody condition, 96 hours).Treatment with antibody #1-25 was statistically significant (p<0.0001)(one way ANOVA and Dunnett's test).

FIG. 42 shows that anti-LSR antibodies enhance p27 expression, suppresscyclin D1 expression, suppress Rb and MAPK activity, and suppress cellgrowth. Expression was observed by Western blot. The left panel shows,from the top, p27, cyclin D1, phosphorylated Rb (retinoblastoma protein;Ser780), phosphorylated Rb (Ser807/811), Rb only, LSR, and GAPDH as acontrol. The right panel shows, from the top, phosphorylated-MEK1/2,MEK1/2, phosphorylated p44/42 MAPK, p44/42 MAPK, and GAPDH. For eachprotein, the results of using, from the left, no treatment, mouse IgG2a,and anti-LSR mAb #1-25 are shown.

FIG. 43 shows that an anti-LSR antibody also has an ADCC non-dependentanti-tumor effect in addition to antitumor effects mediated by ADCC.This is an experiment modeled after RMG-I (NOD/SCID). The graph showsdays after treatment (horizontal axis) and tumor volume (mm³) (verticalaxis). The rhombuses indicate the control IgG (N=6) and the trianglesindicate treatment with anti-LSR Ab (#1-25) (N=6). *, **, and***indicate statistical significance (p<0.05, 0.01, and 0.001, Student'st-test), respectively.

FIG. 44 is another graph showing that an anti-LSR antibody also has ADCCnon-dependent anti-tumor effects in addition to an antitumor effectmediated by ADCC. The left side shows the control IgG administered groupand the right side shows the anti-LSR antibody (#1-25) administeredgroup. Each group was N=6, and the vertical axis is tumor weight (mg).RMG-I (NOD/SCID) was used as the model. The results were statisticallysignificant (p<0.001, Student's t-test).

FIG. 45 shows that tumor cells in the growth phase were decreased invivo by anti-LSR antibodies. Anti-Ki67 antibodies were used forimmunohistochemical staining, and RMG-I (NOD-SCID) was used. The leftcolumn shows the control IgG administered group, and the right columnshows the anti-LSR antibody (#1-25) administered group. The top rowshows 100 times magnification and the bottom row shows 400 timesmagnification.

FIG. 46 shows examination of antitumor effects of anti-LSR antibodies onovarian cancer cell strain (SKOV3-E1, SKOV3-L45, and xenograph model).#1-25 was used as the LSR antibody, and mouse IgG2a (Sigma M7769) wasused as the control. 10 mg/kg was intraperitoneally administered.SKOV3-E1 was used as an empty vector introduced strain, and SKOV3-L45was used as a strain stably expressing LSRs. The arrows on the top sideindicate intraperitoneal administration (every other day, up to day 14),and the bottom indicates tumor volume measurement (every 4 days up today 16 as well as measurement on day 18). An SCID female 6-week oldmouse was used as a model. The tumor size was about 60 mm³ on day 0.

FIG. 47 shows that an anti-LSR monoclonal antibody exhibits an antitumoreffect on an ovarian cancer cell strain xenograft model expressing LSRs.The graph on the left shows SKOV3-L45 (SCID) (strains stably expressingLSRs) and the graph on the right shows SKOV3-E1 (SCID) (empty vector).For each graph, the horizontal axis indicates the days after treatment,and the vertical axis indicates the tumor volume (mm³). The rhombusesindicate the control IgG (N=5) and the triangles indicate treatment withanti-LSR Ab (#1-25) (N=5). *, **, and *** indicate statisticalsignificance (p<0.05, 0.01, and 0.001, Student's t-test), respectively.

FIG. 48 shows that an anti-LSR monoclonal antibody exhibits an antitumoreffect on an ovarian cancer cell strain xenograft model expressing LSRs.The graph on the left shows SKOV3-L45 (SCID) (strains stably expressingLSRs) and the graph on the right shows SKOV3-E1 (SCID) (empty vector).N=5 for each group, and the vertical axis is the tumor weight (mg). Therhombuses indicate the control IgG (N=5) and the triangles indicatetreatment with anti-LSR Ab (#1-25) (N=5). As shown, it was demonstratedthat anti-LSR monoclonal antibodies do not exhibit an antitumor effecton LSR negative cells, but exhibit an antitumor effect specificallyagainst LSR expressing positive cells (was statistically significant(p<0.00076, Student's t-test)).

FIG. 49 shows that an LSR incorporates VLDL and promotes lipidmetabolism. The left side shows a vector introduced cell, and the rightside shows cells forced to express LSRs.

FIG. 50 shows results of examining intracellular incorporation of LSRmonoclonal antibodies. The top row shows results of SKOV3-LSR#45 and thebottom row shows results of SKOV3-Empty#1 (empty vector). The resultsare shown, from the left, for control treatment, Herceptin treatment,antibody #1-25 treatment, and antibody #9-7 treatment. The arrowsindicate various clones of anti-LSR antibodies or Herceptin incorporatedinto the cell.

FIG. 51 shows results of examining intracellular incorporation of LSRmonoclonal antibodies similar to FIG. 50. The top row shows results forSKOV3-LSR#45 and the bottom row shows SKOV3-Empty#1 (empty vector). Fromthe left, antibodies #16-6, #26-2, #27-6 and #1-43 are shown. The arrowsshow various clones of anti-LSR antibodies incorporated into the cell.

FIG. 52 shows the protocol for a safety test on anti-LSR antibodiesusing a mouse. 1 mg/body weight of mouse IgG2a (Sigma M7769) andanti-LSR antibody #1-26 was intraperitoneally administered to C57BL/6J(8 weeks old) to assess the following items on day 7. The brain, heart,kidney, liver, lung and spleen are selected as the extracted organs. Themeasured items include while blood cell (WBC), red blood cell (RBC),hemoglobin (Hb), platelet (Plt), total bilirubin (T-Bil), alanineaminotransferase (ALT), alkaline phosphatase (ALP), amylase (Amy), bloodurea nitrogen (BUN), chrome (Cr), calcium (Ca), phosphorus (P), totalprotein (TP), albumin (Alb), sodium (Na), potassium (K), globulin(Globn), and glutamine (Glu). VetScan™ HMII (Abaxis, Inc.) was used asan automated blood cell counter, and VetScan′ VS2 (Abaxis, Inc. 0) wasused as a veterinary biochemical blood analyzer.

FIG. 53 shows a comparison of control IgG versus anti-LSR antibody(male). In the Table, the left column shows the items, second columnfrom the left shows control IgG (n=3), the third column shows anti-LSRantibodies (n=3), the second column from the right shows normal values,and the right end shows the p value (statistical significance inStudent's t-test). In addition to the abbreviations explain in FIG. 52,Ly indicates lymphocytes and Mo indicates monocytes. Gr indicatesgranulocytes and Hct indicates hematocrit values.

FIG. 54 shows a comparison of control IgG versus anti-LSR antibody(female). Each of the values is the same as that in FIGS. 52-53.

FIG. 55 shows a comparison of control IgG versus anti-LSR antibody(male). In the Table, the left column shows the items, the second columnfrom the left shows control IgG (n=3), the third column shows anti-LSRantibodies (n=3), the second column from the right shows normal values,and the right end shows the p value (statistical significance inStudent's t-test). The abbreviations are as explained in FIG. 52.

FIG. 56 shows a comparison of control IgG versus anti-LSR antibody(female). Each of the values is the same as that in FIGS. 52-53 and 55.

DESCRIPTION OF EMBODIMENTS

The embodiments of the present invention are described in detailhereinafter. It should be noted that descriptions are omitted whenappropriate for the same content in order to avoid complicating thecontent by repeating. Throughout the entire specification, a singularexpression should be understood as encompassing the concept thereof inthe plural form, unless specifically noted otherwise. Thus, singulararticles (e.g., “a”, “an”, “the” and the like in case of English) shouldalso be understood as encompassing the concept thereof in the pluralform unless specifically noted otherwise. Further, the terms used hereinshould be understood as being used in the meaning that is commonly usedin the art, unless specifically noted otherwise. Thus, unless definedotherwise, all terminologies and scientific technical terms that areused herein have the same meaning as the terms commonly understood bythose skilled in the art to which the present invention pertains. Incase of a contradiction, the present specification (including thedefinitions) takes precedence.

First, explanations are provided for the terms and general techniquesused in the present invention.

As used herein, “LSR (lipolysis stimulated lipoprotein receptor)” isgenerally known as a molecule associated with the metabolism of a lowdensity lipoprotein (LDL). The details of the amino acid sequence or thelike of LSRs can be found on the websites of the NCBI (National Centerfor Biotechnology Information), HGNC (HUGO Gene Nomenclature Committee)or the like. Examples of accession numbers of LSRs described in NCBI areNP_991403 (amino acid) and/NM_205834.3 (mRNA). An example of the aminoacid sequence of an LSR is SEQ ID NO: 7. An example of the base sequenceof an LSR mRNA is SEQ ID NO: 8. The amino acid sequence of an LSR is notlimited, as long as the sequence has LSR activity. Thus, it isunderstood that not only proteins (or nucleic acid encoding the same)having an amino acid sequence set forth in a specific sequenceidentification number or accession number, but also a functionallyactive analog or derivative thereof, a functionally active fragmentthereof or homolog thereof, or a mutant encoded by a nucleic acid whichhybridizes to a nucleic encoding said protein under a highly stringentcondition or lowly stringent condition can also be used in the presentinvention, as long as they align with the specific objective of thepresent invention.

As used herein, “derivative”, “analog”, or “mutant” includes, but is notintended to be limited to, molecules comprising a region substantiallyhomologous to a target protein (e.g., LSR). Such a molecule, in variousembodiments, is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99%identical throughout the amino acid sequence of the same size or incomparison to a sequence aligned by a homology computer program known inthe art. Alternatively, a nucleic acid encoding such a molecule canhybridize to a sequence encoding the constituent protein under a(highly) stringent condition, moderately stringent condition, ornon-stringent condition. This refers to a product of altering anaturally-occurring protein by an amino acid substitution, deletion andaddition, respectively, a protein whose derivative exhibits thebiological function of the naturally-occurring protein, although notnecessarily to the same degree. For instance, the biological function ofsuch a protein can be investigated by a suitable and available in vitroassay described herein or known in the art. As used herein,“functionally active” refers to polypeptides, i.e., fragments orderivatives, having a structural function, regulatory function orbiochemical function of a protein such as biological activity inaccordance with an embodiment associated with the polypeptides, i.e.,fragments or derivatives, of the present invention. The discussionregarding LSRs in the present invention mainly pertains to humans, butit is understood that many animals other than humans, especiallymammals, are within the scope of the present invention, as they areknown to express LSRs. Preferably, the functional domain of LSRs e.g.,transmembrane domain (positions 260-280) or phosphorylation sites(positions 309, 328, 406, 493, 528, 530, 535, 540, 551, 586, 615, and646), are conserved.

A fragment of an LSR in the present invention is a polypeptidecomprising any region of the LSR. As long as such a fragment serves thefunction of interest (e.g., marker or therapeutic target) of the presentinvention, it is not necessary that the fragment has biologicalfunctions of a naturally-occurring LSR.

Thus, a representative nucleotide sequence of an LSR may be:

(a) a polynucleotide having a base sequence set forth in SEQ ID NO: 7 ora fragment sequence thereof;

(b) a polynucleotide encoding a polypeptide consisting of the amino acidsequence set forth in SEQ ID NO: 8 or a fragment thereof;

(c) a polypeptide encoding a variant polypeptide having a mutationselected from the group consisting of a substitution, addition, anddeletion of one or more amino acids in the amino acid sequence set forthin SEQ ID NO: 8, the variable polypeptide having biological activity, ora fragment thereof;

(d) a polynucleotide, which is a splice mutant or an allelic mutant ofthe base sequence set forth in SEQ ID NO: 7, or a fragment thereof;

(e) a polynucleotide encoding a species homolog of a polypeptideconsisting of the amino acid sequence set forth in SEQ ID NO: 8, or afragment thereof;

(f) a polynucleotide encoding a polypeptide, which hybridizes with thepolynucleotide of any one of (a)-(e) under stringent conditions and hasbiological activity; or

(g) a polynucleotide encoding a polypeptide consisting of a basesequence, which is at least 70%, at least 80%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto the polynucleotide of any one of (a)-(e) or a complementary sequencethereof and has biological activity. Biological activity in this regardtypically refers to the property of being distinguishable from otherproteins that are present in the same organism as a marker or activityof an LSR. The amino acid of an LSR may be

(a) a polypeptide consisting of the amino acid sequence set forth in SEQID NO: 8 or a fragment thereof;

(b) a polypeptide, which has a mutation selected from the groupconsisting of a substitution, addition, and deletion of one or moreamino acids in the amino acid sequence set forth in SEQ ID NO: 8 and hasbiological activity;

(c) a polypeptide encoded by a splice mutant or an allelic mutant of thebase sequence set forth in SEQ ID NO: 7;

(d) a polypeptide, which is a species homolog of the amino acid sequenceset forth in SEQ ID NO: 8;

(e) a polypeptide, which has an amino acid sequence that is at least70%, at least 80%, at least 90%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% identical to the polypeptide of anyone of (a)-(d) and has biological activity. Biological activity in thisregard typically refers to the property of being distinguishable fromother proteins that are present in the same organism as a marker oractivity of an LSR (for example, when used as an antigen, a property ofcomprising a region that can function as a specific epitope).

In the context of the present invention, “substance that binds to anLSR”, “LSR binding agent”, or “LSR interaction molecule” is a moleculeor substance that binds at least transiently to an LSR. For detectionpurposes, it is preferable that such a molecule or substance isadvantageously capable of indicating that the molecule or substance isbound (i.e., labelled or in a labelable state). For therapeuticpurposes, it is more advantageous that such a molecule or substance isbound to a therapeutic agent. Examples of a substance that binds to anLSR include antibodies, antisense oligonucleotides, siRNAs, lowmolecular weight molecules (LMW), binding peptides, aptamers, ribozymes,peptidomimetics and the like. A substance that binds to an LSR or LSRinteraction molecule may be an LSR inhibitor, and encompasses, forinstance, binding proteins or binding peptides directed to an LSR,especially those directed to an active site of an LSR, as well asnucleic acids directed to a gene of an LSR. A nucleic acid directed toan LSR refers to, for example, a double stranded or single stranded DNAor RNA inhibiting the expression of an LSR gene or activity of an LSR ora modified product or derivative thereof, including, but not limited to,antisense nucleic acids, aptamers, siRNAs (small interfering RNA) andribozymes. As used herein, “binding protein” or “binding peptide”, withrespect to an LSR, refers to any protein or peptide that binds to theLSR, including, but not limited to, antibodies directed to the LSR(e.g., polyclonal antibodies or monoclonal antibodies), antibodyfragments and functional equivalents.

As used herein, “protein”, “polypeptide”, “oligopeptide” and “peptide”are used herein in the same meaning and refer to an amino acid polymerof any length. The polymer may be straight, branched or cyclic. An aminoacid may be a naturally-occurring, non-naturally occurring or alteredamino acid. The term may also encompass those assembled into a complexof multiple polypeptide chains. The term also encompassesnaturally-occurring or artificially altered amino acid polymers.Examples of such an alteration include disulfide bond formation,glycosylation, lipidation, acetylation, phosphorylation, and any othermanipulation or alteration (e.g., conjugation with a labelingcomponent). The definition also encompasses, for example, polypeptidescomprising one or more analogs of an amino acid (e.g., includingnon-naturally occurring amino acids and the like), peptide-likecompounds (e.g., peptoids) and other alterations in the art. As usedherein, “amino acid” is a general term for organic compounds with anamino group and a carboxyl group. When the antibody according to anembodiment of the present invention comprises a “specific amino acidsequence”, any of the amino acids in the amino acid sequence may bechemically modified. Further, any of the amino acids in the amino acidsequence may be forming a salt or a solvate. Further, any of the aminoacids in the amino acid sequence may have an L form or a D form. Evenfor such cases, the protein according to an embodiment of the presentinvention is considered as comprising the above-described “specificamino acid sequence”. Examples of known chemical modifications appliedto an amino acid comprised in a protein in a living body includemodifications of the N-terminus (e.g., acetylation, myristylation andthe like), modifications of the C-terminus (e.g., amidation, addition ofglycosylphosphatidylinositol and the like) modifications of a side chain(e.g., phosphorylation, glycosylation and the like) and the like. Themodifications may be naturally-occurring or non-naturally occurring, aslong as the objective of the present invention is met.

As used herein, “polynucleotide”, “oligonucleotide” and “nucleic acid”are used herein in the same meaning, and refer to a polymer ofnucleotides with any length. The terms also encompass “oligonucleotidederivative” and “polynucleotide derivative”. “Oligonucleotidederivative” and “polynucleotide derivative” refer to an oligonucleotideor polynucleotide that comprises a nucleotide derivative or has a bondbetween nucleotides which is different from normal. The terms are usedinterchangeably. Specific examples of such an oligonucleotide include2′-O-methyl-ribonucleotide, oligonucleotide derivatives having aphosphodiester bond in an oligonucleotide converted to aphosphorothioate bond, oligonucleotide derivatives having aphosphodiester bond in an oligonucleotide converted to an N3′-P5′phosphoramidate bond, oligonucleotide derivatives having ribose andphosphodiester bond in an oligonucleotide converted to a peptide nucleicacid bond, oligonucleotide derivatives having uracil in anoligonucleotide replaced with C-5 propinyluracil, oligonucleotidederivatives having uracil in an oligonucleotide replaced with C-5thiazoluracil, oligonucleotide derivatives having cytosine in anoligonucleotide replaced with C-5 propinylcytosine, oligonucleotidederivatives having cytosine in an oligonucleotide replaced withphenoxazine-modified cytosine, oligonucleotide derivatives having ribosein DNA replaced with 2′-O-propylribose, oligonucleotide derivativeshaving ribose in an oligonucleotide replaced with 2′-methoxyethoxyriboseand the like. Unless noted otherwise, specific nucleic acid sequencesare also intended to encompass conservatively altered variants (e.g.,degenerate codon substitute) and complement sequences as well as theexpressly shown sequences. Specifically, degenerate codon substitutescan be achieved by preparing a sequence with the third position of oneor more selected (or all) codons substituted with a mixed base and/ordeoxyinosine residue (Batzer et al., Nucleic Acid Res. 19: 5081 (1991);Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al.,Mol. Cell. Probes 8: 91-98 (1994)). As used herein, “nucleic acid” isused interchangeably with a gene, cDNA, mRNA, oligonucleotide, andpolynucleotide. As used herein, “nucleotide” may be anaturally-occurring or non-naturally occurring.

As used herein, “gene” refers to an agent defining a genetic trait.“Gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleicacid”.

As used herein, “homology” of genes refers to the level of identity oftwo or more genetic sequences with one another. In general, having“homology” refers to having a high level of identity or similarity.Thus, two genes with high homology have higher identity or similarity ofsequences. It is possible to investigate whether two types of genes arehomologous by direct comparison of sequences or, for nucleic acids, by ahybridization method under a stringent condition. When two geneticsequences are directly compared, the genes are homologous when DNAsequences are representatively at least 50% identical, preferably atleast 70% identical, and more preferably at least 80%, 90%, 95%, 96%,97%, 98%, or 99% identical between the genetic sequences. Thus, as usedherein, “homolog” or “homologous gene product” refers to a protein inanother species, preferably mammal, exerting the same biologicalfunction as a protein constituent of a complex which will be furtherdescribed herein. Such a homolog is also called “ortholog gene product”.It is understood that such a homolog, homologous gene product, orthologgene product or the like can also be used, as long as they are inalignment with the objective of the present invention.

Amino acids may be mentioned herein by either their commonly known threeletter symbols or their one character symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotidesmay be mentioned by their commonly recognized one character codes.Comparison of similarity, identity and homology of an amino acidsequence and abase sequence is calculated herein by using a defaultparameter using a sequence analysis tool, BLAST. For example, identitycan be searched by using BLAST 2.2.28 (published on Apr. 2, 2013) of theNCBI. Herein, values for identity generally refer to a value obtained byalignment under the default condition using the above-described BLAST.However, when a higher value is obtained by changing a parameter, thehighest value is considered the value of identity. When identity isevaluated in a plurality of regions, the highest value thereamong isconsidered the value of identity. Similarity is a value calculated bytaking into consideration a similar amino acid in addition to identity.

In one embodiment of the present invention, “several” may be, forexample, 10, 8, 6, 5, 4, 3 or 2, or a value less than any one of thevalues. It is known that a polypeptide with one or several amino acidresidue deletions, additions, insertions, or substitutions by otheramino acids maintains its biological activity (Mark et al., Proc NatlAcad Sci USA. 1984 September; 81(18): 5662-5666., Zoller et al., NucleicAcids Res. 1982 Oct. 25; 10(20): 6487-6500., Wang et al., Science. 1984Jun. 29; 224 (4656): 1431-1433.). An antibody with a deletion or thelike can be made, for example, by site-directed mutagenesis, randommutagenesis, biopanning using an antibody phage library or the like. Forexample, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used forsite-directed mutagenesis. An antibody with the same activity as thewild-type can be selected from mutant antibodies introduced with adeletion or the like by performing various characterizations such asFACS analysis and ELISA.

In one embodiment of the present invention, “90% or greater” may be, forexample, 90, 95, 96, 97, 98, 99 or 100% or greater or within the rangeof any two values described above. For the above-described “homology”,the percentage of the number of homologous amino acids in two or aplurality of amino acid sequences may be calculated in accordance with aknown method in the art. Before calculating the percentage, amino acidsequences in a group of amino acid sequences to be compared are aligned.A space is introduced in a portion of amino acid sequences whennecessary to maximize the percentage of the same amino acids. Analignment method, method of calculating the percentage, comparisonmethod, and computer programs associated therewith have been well knownin the art (e.g., BLAST, GENETYX and the like). As used herein,“homology” can be represented by a value measured with BLAST of theNCBI, unless specifically noted otherwise. Blastp can be used in thedefault setting for an algorithm for comparing amino acid sequences withBLAST. Results of measurement are expressed in a numerical form asPositives or Identities.

As used herein, “polynucleotide which hybridizes under a stringentcondition” refers to commonly used, well-known conditions in the art.Such a polynucleotide can be obtained by using a method such as colonyhybridization, plaque hybridization, or southern blot hybridizationwhile using a polynucleotide selected from among the polynucleotides ofthe present inventions as a probe. Specifically, the above-describedpolynucleotide refers to a polynucleotide that can be identified byusing a filter with immobilized DNA from a colony or plaque andperforming hybridization at 65° C. in the presence of 0.7-1.0 M NaCl andthen using an SSC (saline-sodium citrate) solution with 0.1-2 timesconcentration (composition of an SSC solution with 1 time concentrationis 150 mM sodium chloride and 15 mM sodium citrate) to wash the filterunder the condition of 65° C. For “stringent condition”, the followingare examples of conditions that can be used. (1) low ionic strength anda high temperature are used for washing (e.g., 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.),(2) a denaturing agent such as formamide is used in hybridization (e.g.,50% (v/v) formamide, 0.1% bovine serum albumin/0.1% ficoll/0.1%polyvinyl pyrrolidone/50 mM sodium phosphate buffer with a pH of 6.5,750 mM sodium chloride, and 75 mM sodium citrate at 42° C.), or (3) asolution comprising 20% formamide, 5×SSC, 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 mg/ml denaturedsheared salmon sperm DNA, is incubated overnight at 37° C. and then afilter is washed with 1×SSC at about 37-50° C. The formamideconcentration may be 50% or greater. Washing time may be 5, 15, 30, 60,120 minutes, or greater. A plurality of elements are considered toaffect stringency in a hybridization reaction such as temperature, saltconcentration and the like. Ausubel et al., Current Protocols inMolecular Biology, Wiley Interscience Publishers, (1995) can be referredfor details. “Highly stringent condition”, for example, is 0.0015 Msodium chloride, 0.0015 M sodium citrate, and 65-68° C. or 0.015 Msodium chloride, 0.0015 M sodium citrate, 50% formamide and 42° C.Hybridization can be performed in accordance with the method describedin experimental publications such as Molecular Cloning 2^(nd) ed.,Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1:Core Techniques, A Practical Approach, Second Edition, Oxford UniversityPress (1995). In this regard, a sequence comprising only an A sequenceor only a T sequence is preferably excluded from a sequence thathybridizes under stringent conditions. A moderately stringent conditioncan be readily determined by those skilled in the art based on, forexample, the length of a DNA and is shown in Sambrook et al., MolecularCloning: A Laboratory Manual, Third Ed., Vol. 1, 7.42-7.45 Cold SpringHarbor Laboratory Press, 2001, including, for a nitrocellulose filters,use of hybridization conditions of a pre-wash solution of 1.0 mM EDTA(pH 8.0), 5×SSC, 0.5% SDS, and about 50% formamide and 2×SSC-6×SSC atabout 40-50° C. (or other similar hybridization solutions such as aStark's solution in about 50% formamide at about 42° C.) and washingconditions of 0.5×SSC, 0.1% SDS at about 60° C. Thus, the polypeptidesused in the present invention encompass polypeptides encoded by anucleic acid molecule that hybridizes under highly or moderatelystringent conditions to a nucleic acid molecule encoding a polypeptidedescribed in the present invention in particular.

As used herein, a “purified” substance or biological agent (e.g.,nucleic acid, protein or the like) refers to a substance or a biologicalagent from which at least a part of an agent naturally accompanying thesubstance or biological agent has been removed. Thus, the purity of abiological agent in a purified biological agent is generally higher thanthe purity in the normal state of the biological agent (i.e.,concentrated). The term “purified” as used herein refers to the presenceof preferably at least 75% by weight, more preferably at least 85% byweight, still more preferably at least 95% by weight, and mostpreferably at least 98% by weight of a biological agent of the sametype. The substance or biological agent used in the present invention ispreferably a “purified” substance. An “isolated” substance or biologicalagent (e.g., nucleic acid, protein, or the like) as used herein refersto a substance or biological agent having agents that naturallyaccompany the substance or biological agent substantially removed. Theterm “isolated” as used herein varies depending on the objective. Thus,the term does not necessarily have to be represented by purity. However,when necessary, the term refers to the presence of preferably at least75% by weight, more preferably at least 85% by weight, still morepreferably at least 95% by weight, and most preferably at least 98% byweight of a biological agent of the same type. The substance used in thepresent invention is preferably an “isolated” substance or biologicalagent.

As used herein, a “corresponding” amino acid, nucleic acid, or moietyrefers to an amino acid or a nucleotide which has or is expected tohave, in a certain polypeptide molecule or polynucleotide molecule(e.g., LSR), similar action as a predetermined amino acid, nucleotide ormoiety in a benchmark polypeptide or a polynucleotide for comparison,and, particularly in the case of enzyme molecules, refers to an aminoacid which is present at a similar position in an active site and makesa similar contribution to catalytic activity and refers to acorresponding moiety in a complex molecule (e.g., transmembrane domainor the like). For example, for an antisense molecule, it can be asimilar moiety in an ortholog corresponding to a specified moiety of theantisense molecule. A corresponding amino acid can be a specified aminoacid subjected to, for example, cysteination, glutathionylation, S—Sbond formation, oxidation (e.g., oxidation of methionine side chain),formylation, acetylation, phosphorylation, glycosylation, myristylationor the like. Alternatively, a corresponding amino acid can be an aminoacid responsible for dimerization. Such a “corresponding” amino acid ornucleic acid may be a region or a domain over a certain range. Thus, itis referred herein as a “corresponding” region or domain in such a case.Such a corresponding region or domain is useful for designing a complexmolecule in the present invention.

As used herein, a “corresponding” gene (e.g., polynucleotide sequence ormolecule) refers to a gene (e.g., polynucleotide sequence or molecule)of a certain species which has or is expected to have similar action asa predetermined gene in a benchmark species for comparison. When thereis a plurality of genes having such action, the corresponding generefers to a gene having the same evolutionary origin. Hence, a genecorresponding to a certain gene may be an ortholog of such a gene. Thus,an LSR corresponding to human LSRs can be found in other animals(especially mammals). Such a corresponding gene can be identified byusing a technique that is well known in the art. For example, acorresponding gene in a certain animal (e.g., mouse) can be found bysearching a database comprising sequences of the animal from using thesequence of SEQ ID NO: 7, 8 or the like as a query sequence, as abenchmark gene of the corresponding gene (e.g., LSR or the like).

As used herein, “fragment” refers to a polypeptide or polynucleotidewith a sequence length of 1 to n-1 with respect to the full lengthpolypeptide or polynucleotide (with length n). The length of a fragmentcan be appropriately changed in accordance with the objective. Examplesof the lower limit of such a length include 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 25, 30, 40, 50 and more amino acids for a polypeptide. Lengthsrepresented by an integer that is not specifically listed herein (e.g.,11 and the like) also can be suitable as a lower limit. Further,examples of length include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,75, 100, and more nucleotides for a polynucleotide. Lengths representedby an integer that is not specifically listed herein (e.g., 11 and thelike) also can be suitable as a lower limit. As used herein, such afragment is understood to be within the scope of the present invention,for example, when a full length version functions as a marker or atarget molecule, as along as the fragment itself also functions as amarker or a target molecule.

The term “activity” according to the present invention refers to afunction of a molecule in the broadest sense herein. Activity, althoughnot intended to be limiting, generally includes a biological function,biochemical function, physical function, and chemical function of amolecule. Examples of activity include enzymatic activity, an ability tointeract with another molecule, an ability to activate, promote,stabilize, inhibit, suppress, or destabilize a function of anothermolecule, stability, and an ability to localize at a specific positionin a cell. When applicable, the term also relates to a function of aprotein complex in the broadest sense.

As used herein, “biological function”, when referring to a certain geneor a nucleic acid molecule or a polypeptide related thereto, refers to aspecific function that the gene, the nucleic acid molecule or thepolypeptide may have in a living body. Examples of such a functioninclude, but are not limited to, production of a specific antibody,enzyme activity, impartation of resistance and the like. In the presentinvention, examples of this function include, but are not limited to, afunction of an LSR involved in inhibition of VLDL incorporation or thelike. As used herein, biological function can be exerted by “biologicalactivity”. As used herein, “biological activity” refers to the activitya certain agent (e.g., polynucleotide, protein or the like) may have ina living body. Biological activity encompasses an activity of exerting avariety of functions (e.g., transcription promoting activity), and alsoencompasses, for example, an activity of activating or inactivatinganother molecule by an interaction with a certain molecule. When twoagents interact, biological activity thereof may be a bond between twomolecules and a biological change induced thereby. For example, twomolecules are considered to be bound together if, when one molecule isprecipitated using an antibody, the other molecule co-precipitates.Observation of such co-precipitation is one example of a determinationapproach. For example, when a certain agent is an enzyme, the biologicalactivity thereof encompasses enzyme activity thereof. In anotherexample, when a certain agent is a ligand, binding to a receptorcorresponding to the ligand is encompassed. Such biological activity canbe measured by a technique that is well known in the art. Thus,“activity” refers to various measurable indicators, which indicate orreveal a bond (either direct or indirect) or affect a response (i.e.,having a measurable effect in response to some exposures of stimuli).Examples thereof includes affinity of a compound that directly binds tothe polypeptide or polynucleotide of the present invention, the amountof proteins upstream or downstream after some stimulations or events, orthe level of other similar functions.

As used herein, “expression” of a gene, a polynucleotide, a polypeptideor the like refers to the gene or the like being subjected to a certainaction in vivo to be converted into another form. Preferably, expressionrefers a gene, a polynucleotide or the like being transcribed andtranslated into a form of a polypeptide. However, transcription to makean mRNA is also one embodiment of expression. Thus, “expression product”as used herein encompasses such a polypeptide or protein, or mRNA. Morepreferably, such a polypeptide form can be a form which has undergonepost-translation processing. For example, the LSR expression level canbe determined by any method. Specifically, the LSR expression level canbe found by assessing the amount of mRNA of LSRs, the amount of LSRprotein, and the biological activity of the LSR protein. The amount ofmRNA or protein of LSRs can be determined by the method described indetail in other parts of the specification or a method known in the art.

As used herein, “functional equivalent” refers to any entity having thesame function of interest but a different structure relative to theoriginal target entity. Thus, it is understood that a functionalequivalent of “LSR” or an antibody thereof encompasses mutants orvariants (e.g., amino acid sequence variant or the like) of the LSR orantibody thereof, not the LSR or antibody thereof itself, which have thebiological action of the LSR and those that can change, upon action,into the LSR or the antibody thereof itself or a mutant or variant ofthe LSR or the antibody thereof (e.g., including nucleic acid encodingan LSR or an antibody thereof itself or a mutant or variant of the LSRor antibody thereof, and vector, cell and the like comprising such anucleic acid). It is understood, even without specific mention, that afunctional equivalent of an LSR or an antibody thereof can be usedsimilarly to the LSR or antibody thereof. A functional equivalent can befound by searching a database or the like. As used herein, “search”refers to utilizing a certain nucleic acid base sequence electronically,biologically, or by another method to find another nucleic acid basesequence having a specific function and/or property. Examples ofelectronic search include, but are not limited to, BLAST (Altschul etal., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc.Natl. Acad. Sci., USA 85: 2444-2448 (1988)), Smith and Waterman method(Smith and Waterman, J. Mol. Biol. 147: 195-197 (1981)), Needleman andWunsch method (Needleman and Wunsch, J. Mol. Biol. 48: 443-453 (1970))and the like. Examples of biological search include, but are not limitedto, stringent hybridization, a macroarray with a genomic DNA applied toa nylon membrane or the like or a microarray with a genomic DNA appliedto a glass plate (microarray assay), PCR, in situ hybridization and thelike. Herein, a gene used in the present invention is intended toinclude corresponding genes identified by such electronic search orbiological search.

As a functional equivalent of the present invention, it is possible touse an amino acid sequence with one or more amino acid insertions,substitutions or deletions, or addition to one or both ends. As usedherein, “one or more amino acid insertions, substitutions or deletions,or addition to one or both ends” in an amino acid sequence refers to analteration with a substitution of a plurality of amino acids or the liketo the extent that can occur naturally by a well-known technical methodsuch as site-directed mutagenesis or natural mutation. An altered aminoacid sequence can have, for example, 1-30, preferably 1-20, morepreferably 1-9, still more preferably 1-5, and especially preferably 1-2amino acid insertions, substitutions or deletions or additions to one orboth ends. Preferably, an altered amino acid sequence may be an aminoacid sequence having one or more (preferably 1 or several, or 1, 2, 3 or4) conservative substitutions in an LSR amino acid sequence.“Conservative substitution” refers herein to a substitution of one ormore amino acid residues with other chemically similar amino acidresidue so as not to substantially alter a function of a protein.Examples thereof include cases where a hydrophobic residue issubstituted with another hydrophobic residue, cases where a polarresidue is substituted with another polar residue having the same chargeand the like. Functionally similar amino acids that can be substitutedin this manner are known in the art for each amino acid. Specificexamples include alanine, valine, isoleucine, leucine, proline,tryptophan, phenylalanine, methionine and the like for nonpolar(hydrophobic) amino acids, glycine, serine, threonine, tyrosine,glutamine, asparagine, cysteine and the like for polar (neutral) aminoacids. Examples of positively charged (basic) amino acid includearginine, histidine, lysine and the like. Further, examples of anegatively-charged (acidic) amino acid include aspartic acid, glutamicacid and the like.

As used herein, “suppressant” refers to a substance or agent thatinhibits biological action of a receptor or a cell against a targetentity (e.g., receptor or cell). An LSR suppressant of the presentinvention is an agent that can temporarily or permanently reduce oreliminate a function of a target LSR, a cell expressing an LSR or thelike. Examples of such a factor include, but are not limited to,antibodies, antigen binding fragments thereof, derivatives, functionalequivalents, antisenses, RNAi agents such as siRNAs and other nucleicacid forms.

As used herein, “agonist” refers to a substance that expresses orenhances biological action of a receptor against a target entity (e.g.,receptor). Examples thereof include natural agonists (also referred toas ligands), as well as synthesized agonists, altered agonists and thelike.

As used herein, “antagonist” refers to a substance that suppresses orinhibits the expression of biological action of a receptor against atarget entity (e.g., receptor). Examples thereof include naturalantagonists (also referred to as ligands), as well as synthesizedantagonists, altered antagonists and the like. Antagonists include thosethat competitively or non-competitively suppress or inhibit expressionagainst an agonist. An antagonist can also be obtained by altering anagonist. Since physiological phenomena are suppressed or inhibited, anantagonist may be encompassed in the concept of suppressant (inhibitor)or suppressing agent. Thus, antagonists as used herein are substantiallyused synonymously with “suppressant”.

As used herein, an “antibody” includes, in a broad sense, polyclonalantibodies, monoclonal antibodies, multi-specific antibodies, chimericantibodies, anti-idiotype antibodies, and fragments thereof such as Fvfragments Fab′ fragments, F(ab′)₂ and Fab fragments, as well as otherconjugates or functional equivalents produced by recombination (e.g.,chimeric antibodies, humanized antibodies, multifunctional antibodies,bispecific or oligospecific antibodies, single chain antibodies, scFV,diabodies, sc(Fv)₂ (single chain (Fv)₂), and scFv-Fc). Furthermore, suchan antibody may be fused, by a covalently bond or recombination, with anenzyme such as alkaline phosphatase, horseradish peroxidase, or agalactosidase. The anti-LSR antibody used in the present invention issufficient if it binds to a protein of LSRs, regardless of the origin,type, shape or the like thereof. Specifically, known antibodies such asa non-human animal antibody (e.g., a mouse antibody, a rat antibody, ora camel antibody), a human antibody, a chimeric antibody, or a humanizedantibody can be used. In the present invention, a monoclonal orpolyclonal antibody can be utilized as an anti-LSR antibody, but amonoclonal antibody is preferable. It is preferable that an antibodybinds specifically to an LSR protein. Further, antibodies encompassmodified and non-modified antibodies. Modified antibodies may be formedby an antibody binding to various molecules such as polyethylene glycol.A modified antibody can be obtained by applying a chemical modificationto an antibody by using a known approach.

“Anti-LSR antibody” in one embodiment of the present inventionencompasses antibodies having binding affinity to LSRs. The productionmethod of such an anti-LSR antibody is not particularly limited. Forexample, the antibody may be produced by immunizing mammals or birdswith an LSR.

Further, it is understood that examples of a “functional equivalent” ofan “antibody to LSR (anti-LSR antibody) or a fragment thereof” includes,for antibodies, antibodies themselves having LSR binding activity andoptionally suppressing activity and fragments thereof themselves, aswell as chimeric antibodies, humanized antibodies, multifunctionalantibodies, bispecific or oligospecific antibodies, single chainantibodies, scFV, diabodies, sc(Fv)₂ (single chain (Fv)₂), scFv-Fc andthe like.

The anti-LSR antibody according to one embodiment of the presentinvention is preferably an anti-LSR antibody that specifically binds toa specific epitope of an LSR from the viewpoint of malignant tumorgrowth being particularly highly suppressed.

The anti-LSR antibody according to one embodiment of the presentinvention may be a monoclonal antibody. A monoclonal antibody can bemade to more efficiently act against an LSR relative to a polyclonalantibody. It is preferred that a chicken is immunized with an LSR fromthe viewpoint of efficient production of anti-LSR monoclonal antibodies.

The antibody class of the anti-LSR antibody according to one embodimentof the present invention is not particularly limited. For example, theclass may be IgM, IgD, IgG, IgA, IgE, or IgY.

The anti-LSR antibody according to one embodiment of the presentinvention may be an antibody fragment having antigen binding activity(hereinafter, also referred to as “antigen binding fragment”). In such acase, there is an effect of improved stability, antibody productionefficiency or the like.

The anti-LSR antibody according to one embodiment of the presentinvention may be a fusion protein. The fusion protein may comprise apolypeptide or oligopeptide bound to the N or C-terminus of an anti-LSRantibody. The oligopeptide in this regard may be an His-tag. The fusionprotein may also be fused to a mouse, human, or chicken antibody partialsequence. Such fusion proteins are also encompassed as one form of theanti-LSR antibody according to the present embodiment.

The anti-LSR antibody according to one embodiment of the presentinvention may be, for example, an antibody obtained via the step ofimmunizing an organism with a purified LSR, LSR-expressing cell, or anLSR containing lipid membrane. It is preferable that an LSR-expressingcell is used for immunization from the viewpoint of enhancing atherapeutic effect against LSR positive malignant tumor.

The anti-LSR antibody according to one embodiment of the presentinvention may be an antibody having a CDR set of an antibody obtainedvia the step of immunizing an organism with a purified LSR,LSR-expressing cell, or an LSR containing lipid membrane. It ispreferable that an LSR-expressing cell is used for immunization from theviewpoint of enhancing a therapeutic effect against LSR positivemalignant tumor. A CDR set is a set of heavy chain CDRs 1, 2, and 3 andlight chain CDRs 1, 2, and 3.

“LSR expressing cell” in one embodiment of the present invention may beobtained, for example, by introducing a polynucleotide encoding an LSRinto a cell and having the LSR expressed. LSRs in this regard encompassLSR fragments. Further, “LSR-containing lipid membrane” in oneembodiment of the present invention may be obtained, for example, bymixing an LSR and a lipid bilayer. LSRs in this regard encompass LSRfragments. Further, the anti-LSR antibody according to one embodiment ofthe present invention is preferably an antibody obtained via the step ofimmunizing a chicken with an antigen or an antibody having a CDR set ofsuch an antibody from the viewpoint of enhancing a therapeutic effectagainst LSR positive malignant tumor.

The anti-LSR antibody according to one embodiment of the presentinvention may have any binding strength as long as the object can beaccomplished. Examples thereof include, but are not limited to, at least1.0×10⁶ or greater, 2.0×10⁶ or greater, 5.0×10⁶ or greater, and 1.0×10⁷or greater. The K_(D) value (kd/ka) generally may be 1.0×10⁻⁷ (M) orless and can be 1.0×10⁻⁹ (M) or 1.0×10⁻¹° or less.

The anti-LSR antibody according to one embodiment of the presentinvention may have ADCC or CDC activity.

The anti-LSR antibody according to one embodiment of the presentinvention may be an antibody that binds to a wild-type or mutant LSR.Mutant LSRs include mutants due to a difference in the DNA sequencesamong individuals. The amino acid sequence of a wild-type or mutant LSRis preferably 80% or more, more preferably 90% or more, more preferably95% or more, and especially preferably 98% or more homologous to theamino acid sequence set forth in SEQ ID NO: 8.

“Antibody” in one embodiment of the present invention encompassesmolecules capable of specifically binding to a specific epitope on anantigen and populations thereof. Further, the antibody may be apolyclonal antibody or monoclonal antibody. The antibody can be presentin various forms. For example, the antibody may be in one or more typesof forms selected from the group consisting of a full-length antibody(antibody having an Fab region and an Fc region), Fv antibody, Fabantibody, F(ab′)₂ antibody, Fab′ antibody, diabody, single chainantibody (e.g., scFv), dsFv, multi-specific antibody (e.g., bispecificantibody), peptide or polypeptide having antigen binding affinity,chimeric antibody (e.g., mouse-human chimeric antibody, chicken-humanchimeric antibody or the like), mouse antibody, chicken antibody,humanized antibody, human antibody, and similar antibodies (orequivalents). Further, the antibody encompasses modified or non-modifiedantibodies. Modified antibodies may be formed by an antibody binding tovarious molecules such as polyethylene glycol. A modified antibody canbe obtained by applying a chemical modification to an antibody by usinga known approach. Furthermore, such an antibody may be fused by acovalent bond or recombination with an enzyme such as alkalinephosphatase, horseradish peroxidase, or a galactosidase. The anti-LSRantibody used in the present invention is sufficient if it binds to anLSR protein, regardless of the origin, type, shape or the like thereof.Specifically, known antibodies such as a non-human animal antibody(e.g., a mouse antibody, a rat antibody, or a camel antibody), a humanantibody, a chimeric antibody, or a humanized antibody can be used. Inthe present invention, a monoclonal or polyclonal antibody can beutilized as the anti-LSR antibody, but a monoclonal antibody ispreferable. It is preferable that an antibody binds specifically to anLSR protein. Further, the antibody encompasses modified and non-modifiedantibodies. Modified antibodies may be formed by an antibody binding tovarious molecules such as polyethylene glycol. A modified antibody canbe obtained by applying a chemical modification to an antibody by usinga known approach.

“Polyclonal antibody” in one embodiment of the present invention can beproduced, for example, by administering an immunogen comprising anantigen of interest to mammals (e.g., rat, mouse, rabbit, cow, monkey orthe like), birds or the like in order to induce production of apolyclonal antibody specific to the antigen. An immunogen may beadministered by one or more immunizing agents and, when desired, aninjection of an adjuvant. An adjuvant may be used to increase immuneresponses and may comprise a Freund's adjuvant (complete or incomplete),mineral gel (aluminum hydroxide or the like), surfactant (lysolecithinor the like) or the like. Immunization protocols are known in the artand, in some cases, may be implemented by any method that induces animmune response, which matches the selected host organism (TanpakushitsuJikken Handobukku [Protein experiment handbook], Yodosha (2003): 86-91).

“Monoclonal antibody” in one embodiment of the present inventionencompasses individual antibodies constituting a population beingantibodies corresponding to substantially a single epitope except forantibodies having a mutation that can occur naturally in small amounts.Further individual antibodies constituting a population may beantibodies that are substantially the same except for antibodies havinga mutation that can occur naturally in small amounts. Monoclonalantibodies are highly specific, which are different from commonpolyclonal antibodies that typically include different antibodiescorresponding to different epitopes. In addition to their specificity,monoclonal antibodies are useful in that they can be synthesized fromhybridoma culture which is not contaminated with other immunoglobulins.The description “monoclonal” may indicate a characteristic of beingobtained from a substantially homogeneous antibody population. However,such a description does not mean that antibodies must be produced by aspecific method. For example, monoclonal antibodies may be made by amethod similar to a hybridoma method as described in “Kohler G, MilsteinC., Nature. 1975 Aug. 7; 256 (5517): 495-497”. Alternatively, monoclonalantibodies may be made by a method similar to a recombinant method asdescribed in U.S. Pat. No. 4,816,567. Further, monoclonal antibodies maybe isolated from a phage antibody library using a method similar to thetechnique that is described in, “Clackson et al., Nature. 1991 Aug. 15;352 (6336): 624-628.” or “Marks et al., J Mol Biol. 1991 Dec. 5; 222(3): 581-597”. Further, monoclonal antibodies may be made by the methoddescribed in “Tanpakushitsu Jikken Handobukku [Protein experimenthandbook], Yodosha (2003): 92-96”.

Antibodies can be mass-produced by using any approach that is known inthe art. Examples of construction of mass production system for arepresentative antibody and antibody manufacture include the following.Specifically, an H chain antibody expression vector and L chain antibodyexpression vector are transfected into a CHO cell. The cells arecultured by using a selection reagent G418 and Zeocin and cloned bylimiting dilution. After cloning, clones stably expressing antibodiesare selected by ELISA. The culture is expanded with selected CHO cells,and the culture supernatant comprising antibodies are collected.Antibodies can be purified from the collected culture supernatant byProtein A or Protein G purification.

“Fv antibody” in one embodiment of the present invention is an antibodycomprising an antigen recognition site. This region comprises a dimer ofone heavy chain variable domain non-covalently bound to one light chainvariable domain. In this configuration, three CDRs of each variabledomain can interact with one another to form an antigen binding site onthe surface of a VH-VL dimer.

“Fab antibody” in one embodiment of the present invention is, forexample, a fragment obtained by treating an antibody comprising an Fabregion and an Fc region with proteinase papain, which is an antibody inwhich about half of the N-terminus side of the H chain is bound to theentire L chain via some disulfide bonds. Fabs can be obtained, forexample, by treating the anti-LSR antibody according to the embodimentsof the present invention comprising an Fab region and an Fc region withproteinase papain.

“F(ab′)₂ antibody” in one embodiment of the present invention is afragment obtained by treating an antibody comprising an Fab region andan Fc region with proteinase pepsin, which is an antibody comprising twosites corresponding to Fabs. F(ab′)₂ can be obtained, for example, bytreating the anti-LSR antibody according to the embodiments of thepresent invention comprising an Fab region and an Fc region withproteinase pepsin. For example, the following Fab′ can be made bythioether bond or a disulfide bond.

“Fab′ antibody” in one embodiment of the present invention is anantibody obtained, for example, by cleaving a disulfide bond of a hingeregion of F(ab′)₂. For example, F(ab′)₂ can be obtained throughtreatment with a reducing agent dithiothreitol.

“ScFv antibody” in one embodiment of the present invention is anantibody comprising VH and VL linked with a suitable peptide linker.ScFv antibodies can be produced, for example, by obtaining a cDNAencoding VH and VL of the anti-LSR antibody according to the embodimentof the present invention, constructing a polynucleotide encodingVH-peptide linker-VL, incorporating the polynucleotide into a vector,and using a cell for expression.

“Diabody” in one embodiment of the present invention is an antibodyhaving divalent antigen binding activity. Divalent antigen bindingactivity can be configured to be identical or configured such that oneof them has a different antigen binding activity. A diabody can beproduced, for example, by constructing a polynucleotide encoding scFvsuch that the length of the amino acid sequence of a peptide linker is 8residues or less, incorporating the resulting polynucleotide into avector and using a cell for expression.

“dsFv” in one embodiment of the present invention is an antibody inwhich a polypeptide introduced with cysteine residues in VH and VL isbound via a disulfide bond between the above-described cysteineresidues. The position to which cysteine residues are introduced can beselected based on steric structure prediction of an antibody inaccordance with the method demonstrated by Reiter et al (Reiter et al.,Protein Eng. 1994 May; 7(5): 697-704).

“Peptide or polypeptide with antigen binding affinity” in one embodimentof the present invention is an antibody comprised of antibody VH, VL orCD1, 2 or 3 thereof. A peptide comprising a plurality of CDRs can bebound directly or via a suitable peptide linker.

The production method of the above-described Fv antibody, Fab antibody,F(ab′)₂ antibody, Fab′ antibody, scFv antibody, diabody, dsFv antibody,and peptide or polypeptide having antigen binding affinity (hereinafter,also referred to as “Fv antibodies”) is not particularly limited. Fvantibodies can be produced, for example, by incorporating a DNA encodinga region of the Fv antibodies in the anti-LSR antibody according to theembodiment of the present invention into an expression vector and usingan expression cell. Further, Fv antibodies may be produced by a chemicalsynthesis method such as the Fmoc (fluorenylmethyloxycarbonyl) or tBOC(t-butyloxycarbonyl) method. It should be noted that the antigen bindingfragment according to one embodiment of the present invention may be oneor more types of the above-described Fv antibodies.

“Chimeric antibody” in one embodiment of the present invention is, forexample, a variable region of an antibody linked to a constant region ofan antibody between xenogenic organisms and can be constructed by agenetic engineering technique. A mouse-human chimeric antibody can bemade by, for example, the method described in “Roguska et al., Proc NatlAcad Sci USA. 1994 Feb. 1; 91 (3): 969-973.” For example, the basicmethod of making a mouse-human chimeric antibody links a mouse leadersequence and a variable region sequence in a cloned cDNA with a sequenceencoding a human antibody constant region already present in anexpression vector of a mammalian cell. Further, after linking the mouseleader sequence and variable region sequence in a cloned cDNA with thesequence encoding a human antibody constant region, the resultantsequence may be linked with a mammalian cell expression vector. Afragment of a human antibody constant region can be from any humanantibody H chain constant region and human antibody L chain constantregion. Examples of human H chain fragment include Cγ1, Cγ2, Cγ3, andCγ4, and examples of L chain fragment include Cλ and Cκ.

“Humanized antibody” in one embodiment of the present invention is, forexample, an antibody, which has one or more CDRs from non-human species,a framework region (FR) from a human immunoglobulin, and a constantregion from human immunoglobulin and binds to a desired antigen.Antibodies can be humanized by using various approaches known in the art(Almagro et al., Front Biosci. 2008 Jan. 1; 13: 1619-1633). Examplesthereof include CDR grafting (Ozaki et al., Blood. 1999 Jun. 1; 93 (11):3922-3930.), Re-surfacing (Roguska et al., Proc Natl Acad Sci USA. 1994Feb. 1; 91 (3): 969-973.), FR shuffle (Damschroder et al., Mol Immunol.2007 April; 44(11): 3049-3060. Epub 2007 Jan. 22.) and the like. Anamino acid residue of a human FR region may be substituted with acorresponding residue from a CDR donor antibody in order to alter(preferably in order to improve) the antigen bond. The FR substitutioncan be implemented by a method well known in the art (Riechmann et al.,Nature. 1988 Mar. 24; 332 (6162):323-327.) For example, an FR residuethat is important for antigen binding may be identified by modeling aninteraction between a CDR and an FR residue. Further, an abnormal FRresidue at a specific position may be identified by sequence comparison.

“Human antibody” in one embodiment of the present invention is, forexample, an antibody in which a region comprising a variable region andconstant region of a heavy chain and variable region and constant regionof a light chain constituting the antibody is derived from a geneencoding a human immunoglobulin. Main production methods include amethod using a transgenic mouse for making human antibodies, phagedisplay and the like. A method using a transgenic mouse for making humanantibodies produces human antibodies with diverse antigen bindingcapabilities instead of mouse antibodies when a functional human Ig geneis introduced into an endogenous Ig knockout mouse. Furthermore, thismouse can be immunized to obtain human monoclonal antibodies by aconventional hybridoma method. Such antibodies can be made, for example,by the method described in “Lonberg et al., Int Rev Immunol. 1995;13(1): 65-93.” Phase display is a system that typically expresses anexogenous gene as a fusion protein such that phage infectivity is notlost on the N-terminus side of a coat protein (g3p, g10p, or the like)of fibrous phage such as M13 or T7 which is an E. coli virus. Antibodiescan be made, for example, by the method described in “Vaughan et al.,Nat Biotechnol. 1996 March; 14(3): 309-314”.

Further, antibodies may be prepared by grafting a heavy chain CDR orlight chain CDR of the anti-LSR antibody according to the embodiment ofthe present invention onto any antibody by CDR-grafting (Ozaki et al.,Blood. 1999 Jun. 1; 93(11): 3922-3930). Further, antibodies can beobtained by linking a DNA encoding a heavy chain CDR or light chain CDRof the anti-LSR antibody according to the embodiment of the presentinvention and a DNA encoding a region excluding a heavy chain CDR orlight chain CDR of a known antibody derived from a human or a non-humanorganism to a vector in accordance with a known method in the art andusing a known cell for expression. When obtaining antibodies in thismanner, a known method in the art (e.g., method of allowing amino acidresidues of an antibody to randomly mutate and screening for antibodieswith high reactivity, phage display, or the like) may be used tooptimize the region excluding a heavy chain CDR or light chain CDR inorder to enhance the efficiency of anti-LSR antibody acting upon atarget antigen. Further, an FR region may be optimized by using, forexample, FR shuffle (Damschroder et al., Mol Immunol. 2007 April; 44(11): 3049-3060. Epub 2007 Jan. 22.) or a method of replacing a vernierzone amino acid residue or packaging residue (Japanese Laid-OpenPublication No. 2006-241026 or Foote et al., J Mol Biol. 1992 Mar. 20;224(2): 487-499).

“Heavy chain” in one embodiment of the present invention is typicallythe main constituent element of a full-length antibody. A heavy chain isgenerally bound to a light chain by a disulfide bond and non-covalentbond. A region called a variable region (VH) which has an amino acidsequence that is not constant even among antibodies in the same class ofthe same species, is present in a domain on the N-terminus side of aheavy chain. VH is generally known to greatly contribute to thespecificity and affinity to an antigen. For example, “Reiter et al., JMol Biol. 1999 Jul. 16; 290 (3): 685-98.” describes that a molecule withonly a VH, when made, bound to an antigen with specificity and highlevel of affinity. Furthermore, “Wolfson W, Chem Biol. 2006 December; 13(12): 1243-1244.” describes that there are antibodies having only aheavy chain without a light chain among camel antibodies.

“CDR (complementarity determining region)” in one embodiment of thepresent invention is a region that is in actual contact with an antigento form a binding site in an antibody. CDRs are generally located on anFv (variable region: including heavy chain variable region (VH) andlight chain variable region (VL)) of an antibody. Further, CDRsgenerally have CDR1, CDR2, and CDR3 consisting of about 5-30 amino acidresidues. In addition, CDRs of a heavy chain are particularly known fortheir contribution to binding of an antibody to an antigen. Among theCDRs, CDR3 is known to contribute the most in binding of an antibody toan antigen. For example, “Willy et al., Biochemical and BiophysicalResearch Communications Volume 356, Issue 1, 27 Apr. 2007, Pages124-128” describes that a heavy chain CDR3 was altered to elevate thebinding capability of an antibody. An Fv region other than the CDRs iscalled a framework region (FR), consisting of FR1, FR2, FR3, and FR4,which are conserved relatively well among antibodies (Kabat et al.,“Sequence of Proteins of Immunological Interest” US Dept. Health andHuman Services, 1983.) Specifically, a factor characterizing thereactivity of an antibody is considered to be in CDRs, especially heavychain CDRs.

A plurality of methods for defining CDRs and determining the positionsthereof have been reported. For example, the Kabat definition (Sequencesof Proteins of Immunological Interest, 5th ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) or the Chothiadefinition (Chothia et al., J. Mol. Biol., 1987; 196: 901-917) may beused. One embodiment of the present invention uses the Kabat definitionas an optimal example, but the definition is not necessarily limitedthereto. Further, the definitions may be determined in some cases afterconsidering both the Kabat definition and the Chonthia definition. Forexample, an overlapping portion of CDR according to each of thedefinitions, or a portion comprising both CDRs according to each of thedefinitions can be deemed the CDR. A specific example of such a methodis the method of Martin et al using Oxford Molecular's AbM antibodymodeling software, which is a proposal combining the Kabat definitionand the Chonthia definition (Proc. Natl. Acad. Sci. USA, 1989; 86:9268-9272). Such CDR information can be used to produce a mutant thatcan be used in the present invention. Such an antibody mutant comprisesone or several (e.g., 2, 3, 4, 5, 6, 7, 8, 9, and 10) substitutions,additions, or deletions in the framework of the original antibody.However, a mutant can be produced such that the CDR does not comprise amutation.

As used herein, “antigen” refers to any substrate that can bespecifically bound by an antibody molecule. As used herein, “immunogen”refers to an antigen that can initiate lymphocyte activation which leadsto an antigen specific immune response. As used herein, “epitope” or“antigen determinant” refers to a site in an antigen molecule to whichan antibody or a lymphocyte receptor binds. A method of determining anepitope is well known in the art. Such an epitope can be determined bythose skilled in the art by using a well-known and conventionaltechnique when a primary sequence of an amino acid or a nucleic acid isprovided. It is understood that the antibody of the present inventioncan be similarly used even for antibodies having other sequences, aslong as the epitope is the same.

It is understood that antibodies with any specificity may be used as theantibody used herein, as long as false positive reactions are reduced.Thus, the antibodies used in the present invention may be a polyclonalantibody or a monoclonal antibody.

As used herein, “means” refers to anything that can be a tool foraccomplishing an objective (e.g., detection, diagnosis, therapy). Asused herein, “selective recognizing means” in particular refers to meanscapable of recognizing a certain subject differently from others.

As used herein, “marker (substance, protein or gene)” refers to asubstance that can be an indicator for tracking whether a target is inor at risk of being in a certain condition (e.g., diseased state,disorder state, level of or presence of malignant state or the like).Examples of such a marker include genes, gene products, metabolites,enzymes and the like. In the present invention, detection, diagnosis,prognosis, poor prognosis, diagnosis of poor prognosis, diagnosis ofprognostic state, preliminary detection, prediction, or prediagnosis ofa certain state (e.g., state of a disease such as cancer) can bematerialized by using an agent or means specific to a marker associatedwith such a state, or a composition, kit or system comprising the sameor the like. As used herein, “expression product” (also referred to as agene product) refers to a protein or mRNA encoded by a gene. It is foundin the present specification that a gene product (LSR), which does notexhibit association with malignant tumor, especially to therapy thereof,can be used as an indicator for ovarian cancer.

“Malignant tumor” as used herein includes, for example, tumor thatdevelops from a mutation of normal cells. Malignant tumor can developfrom any organ or tissue of the entire body. Such malignant tumorcomprises one or more type selected from the group consisting of lungcancer, esophageal cancer, gastric cancer, liver cancer, pancreaticcancer, renal cancer, adrenal cancer, bile duct cancer, breast cancer,colon cancer, small intestine cancer, ovarian cancer, uterine cancer,bladder cancer, prostate cancer, ureteral cancer, renal pelvis cancer,ureteral cancer, penile cancer, testicular cancer, cerebral tumor,cancer of the central nervous system, cancer of the peripheral nervoussystem, head and neck cancer, glioma, glioblastoma multiform, skincancer, melanoma, thyroid cancer, salivary gland cancer, malignantlymphoma, carcinoma, sarcoma, and hematologic malignancy. Ovarian cancerin this regard includes, for example, ovarian serous adenocarcinoma andovarian clear cell adenocarcinoma. Uterine cancer includes, for example,endometrial cancer and cervical cancer. Head and neck cancer includes,for example, oral cavity cancer, pharyngeal cancer, nasal cavity cancer,paranasal cancer, salivary gland cancer, and thyroid cancer. Lung cancerincludes, for example, non-small-cell lung cancer and small cell lungcancer. Further, malignant tumor may be LSR positive.

Among malignant tumor, serous adenocarcinoma is cancer that progressesvery rapidly. It is difficult to completely eliminate the cancer evenwith commercially available anticancer agents. Furthermore, inrecurrences, commercially available anticancer agents hardly have anyeffect thereon. Further, hardly any therapeutic effect can be expectedon clear cell adenocarcinoma by commercially available anticanceragents. Meanwhile, the anti-LSR antibody according to the embodiment ofthe present invention can be a novel therapeutic drug for serousadenocarcinoma and clear cell adenocarcinoma.

“LSR positive malignant tumor” in one embodiment of the presentinvention includes malignant tumor that significantly expresses oroverexpresses LSRs. Whether malignant tumor is LSR positive may beassessed, for example, by RT-PCR, Western blot, or immunohistochemicallystaining method. Further, when total protein of malignant tumor cells issubjected to Western blot and a band corresponding to LSRs (e.g., bandnear 649aa) can be observed by visual inspection, the malignant tumormay be determined as LSR positive. Further, when the amount of LSRexpression of malignant tumor cells from a patient is significantly morethan for normal cells, the malignant tumor may be determined as LSRpositive. It is preferable that an anti-LSR antibody is used to inspectLSR expression from the viewpoint of materializing a more optimal dosingby accurately diagnosing malignant tumor as LSR positive.

As used herein, “subject (person)” refers to a target subjected todiagnosis, detection, therapy or the like of the present invention(e.g., an organism such as a human or a cell, blood, serum or the likeextracted from an organism).

As used herein, “sample” refers to any substance obtained from a subjector the like. For example, serum and the like are encompassed thereby.Those skilled in the art can appropriately select a preferred samplebased on the descriptions herein.

As used herein, “agent” is used broadly and may be any substance orother elements (e.g., energy, radiation, heat, electricity and otherforms of energy) as long as the intended objective can be achieved.Examples of such a substance include, but are not limited to, protein,polypeptide, oligopeptide, peptide, polynucleotide, oligonucleotide,nucleotide, nucleic acid (including, for example, DNAs such as cDNA andgenomic DNA and RNAs such as mRNA), polysaccharide, oligosaccharide,lipid, organic small molecule (e.g., hormone, ligand, informationtransmitting substance, organic small molecule, molecule synthesized bycombinatorial chemistry, small molecule that can be used as medicine(e.g., small molecule ligand and the like)) and a complex moleculethereof. Typical examples of an agent specific to a polynucleotideinclude, but are not limited to, a polynucleotide having complementaritywith a certain sequence homology (e.g., 70% or greater sequenceidentity) to a sequence of the polynucleotide, polypeptide such as atranscription factor that binds to a promoter region and the like.Typical examples of an agent specific to a polypeptide include, but arenot limited to, an antibody directed specifically to the polypeptide ora derivative or analog thereof (e.g., single stranded antibody), aspecific ligand or receptor when the polypeptide is a receptor orligand, a substrate when the polypeptide is an enzyme and the like.

As used herein, “diagnosis” refers to identifying various parametersassociated with a disease, disorder, condition (e.g., malignant tumor)or the like in a subject to determine the current or future state ofsuch a disease, disorder, or condition. The condition in the body can beinvestigated by using the method, apparatus, or system of the presentinvention. Such information can be used to select and determine variousparameters of a formulation or method for the treatment or prevention tobe administered, disease, disorder, or condition in a subject or thelike. As used herein, “diagnosis” when narrowly defined refers todiagnosis of the current state, but when broadly defined includes “earlydiagnosis”, “predictive diagnosis”, “prediagnosis” and the like. Sincethe diagnostic method of the present invention in principle can utilizewhat comes out from a body and can be conducted away from a medicalpractitioner such as a physician, the present invention is industriallyuseful. In order to clarify that the method can be conducted away from amedical practitioner such as a physician, the term as used herein may beparticularly called “assisting” “predictive diagnosis, prediagnosis ordiagnosis”.

The term “prognosis” as used herein refers to prediction of thepossibility of progression or death due to cancer, such as thepossibility of recurrence, metastasis and diffusion, drug resistance andthe like of a neoplastic disease such as malignant tumor (e.g., ovariancancer). Thus, as used herein, “excellent prognostic state” refers to astate where recurrence of primary cancer is not observed beyond acertain period of time (e.g., 4 years) after removal of the cancertissue, and “poor prognostic state” or “poor prognosis” refers to astate where recurrence of primary cancer is observed beyond a certainperiod of time (e.g., 4 years) after removal of the cancer tissue. Aprognostic agent is a variable related to natural course of malignanttumor, which affects the rate of recurrence of outcome of a patient whohas experienced malignant tumor. Examples of clinical indicatorassociated with exacerbation in prognosis include lymph node metastasisand highly malignant tumor. A prognostic agent is often used to classifypatients into subgroups with different fundamental risks of recurrence.In this manner, expression of LSRs of the present invention can be usedas a prognostic agent. The term “prediction” as used herein refers tothe possibility of a patient having a specific clinical outcome,regardless of good or bad, after extraction of primary tumor. Thus, LSRsof the present invention can be used as a poor prognosis marker. Atherapeutic method can be determined by clinically using the predictionmethod of the present invention to select the optimal therapeutic methodfor a specific patient. The prediction method of the present inventionwould be a beneficial means for prediction if there is a possibilitythat a patient would have a positive reaction to a therapeutic regimen,e.g., surgical intervention or the like. A prognostic agent can beincluded in the prediction.

As used herein, “detecting drug (agent)” or “inspection drug (agent)”broadly refers to all agents capable of detecting or inspecting a targetof interest.

As used herein, “diagnostic drug (agent)” broadly refers to all agentscapable of diagnosing a condition of interest (e.g., disease such asmalignant tumor or the like).

As used herein, “therapy” refers to the prevention of exacerbation,preferably maintaining of the current condition, more preferablyalleviation, and still more preferably disappearance of a disease ordisorder (e.g., malignant tumor) in case of such a condition, includingbeing capable of exerting a prophylactic effect or an effect ofimproving a disease of a patient or one or more symptoms accompanyingthe disease. Preliminary diagnosis with suitable therapy may be referredto as “companion therapy” and a diagnostic agent therefor may bereferred to as “companion diagnostic agent”.

As used herein, “therapeutic drug (agent)” broadly refers to all agentscapable of treating a condition of interest (e.g., diseases such asmalignant tumor or the like). In one embodiment of the presentinvention, “therapeutic drug” may be a pharmaceutical compositioncomprising an effective ingredient and one or more pharmacologicallyacceptable carriers. A pharmaceutical composition can be manufactured,for example, by mixing an effective ingredient and the above-describedcarriers by any method known in the technical field of pharmaceuticals.Further, mode of usage of a therapeutic drug is not limited, as long asit is used for therapy. A therapeutic drug may be an effectiveingredient alone or a mixture of an effective ingredient and anyingredient. Further, the shape of the above-described carriers is notparticularly limited. For example, the carrier may be a solid or liquid(e.g., buffer solution). It should be noted that a therapeutic drug ofmalignant tumor includes a drug (prophylactic drug) for preventingmalignant tumor or a growth suppressant for malignant tumor cells.

As used herein, “prevention” refers to the action of taking a measureagainst a disease or disorder (e.g., malignant tumor) from being in sucha condition prior to being in such a condition. For example, it ispossible to use the agent of the present invention to perform diagnosis,and optionally use the agent of the present invention to prevent or takemeasures to prevent malignant tumor or the like.

As used herein, “prophylactic drug (agent)” broadly refers to all agentscapable of preventing a condition of interest (e.g., diseases such asmalignant tumor or the like).

As used herein, “interaction” refers, for two substances, to applying aforce (e.g., intermolecular force (Van der Waals force), hydrogen bond,hydrophobic interaction, or the like) between one substance and theother substance. Generally, two substances that have interacted are in aconjugated or bound state. The detection, inspection, and diagnosis inthe present invention can be materialized by utilizing such interaction.

As used herein, the term “bond” refers to a physical or chemicalinteraction between two substances or between combinations thereof. Abond includes an ionic bond, non-ionic bond, hydrogen bond, Van derWaals bond, hydrophobic interaction and the like. A physical interaction(bond) may be direct or indirect. Indirect physical interaction (bond)is mediated by or is due to an effect of another protein or compound. Adirect bond refers to an interaction, which does not occur through ordue to an effect of another protein or compound and does notsubstantially involve another intermediate.

Thus, an “agent” (or detection agent or the like) that “specifically”interacts (or binds) to a biological agent such as a polynucleotide or apolypeptide as used herein encompasses agents with affinity to thebiological agent such as a polynucleotide or polypeptide that istypically similar or higher, preferably significantly (e.g.,statistically significantly) higher, than affinity to other unrelatedpolynucleotides or polypeptides (especially those with less than 30%identity). Such affinity can be measured, for example, by hybridizationassay, binding assay or the like.

As used herein, “specific” interaction (or bond) of a first substance oragent with a second substance or agent refers to the first substance oragent interacting with (or binding to) the second substance or agent ata higher level of affinity than to substances or agents other than thesecond substance or agent (especially other substances or agents in asample comprising the second substance or agent). Examples of aninteraction (or bond) specific to a substance or agent include, but arenot limited to, hybridization in a nucleic acid, antigen-antibodyreaction in a protein, enzyme-substrate reaction, other nucleic acidprotein reactions, protein-lipid interaction, nucleic acid-lipidinteraction and the like. Thus, when substances or agents are bothnucleic acids, a first substance or agent “specifically interacting”with a second substance or agent encompasses the first substance oragent having at least partial complementarity to the second substance oragent. Further, examples of a first substance or agent “specifically”interacting with (or binding to) a second substance or agent whensubstances or agents are both proteins include, but are not limited to,interaction by an antigen-antibody reaction, interaction by areceptor-ligand reaction, enzyme-substrate interaction and the like.When two types of substances or agents include a protein and a nucleicacid, a first substance or agent “specifically” interacting with (orbinding to) a second substance or factor encompasses an interaction (ora bond) between an antibody and an antigen. Such a specific interactiveor binding reaction can be utilized to detect or quantify a target in asample.

As used herein, “detection” or “quantification” of polynucleotide orpolypeptide expression can be accomplished by using a suitable methodincluding, for example, an immunological measuring method andmeasurement of mRNAs, including a bond or interaction to a detectionagent, inspection agent or diagnostic agent. Examples of a molecularbiological measuring method include northern blot, dot blot, PCR and thelike. Examples of an immunological measurement method include ELISAusing a microtiter plate, RIA, fluorescent antibody method, luminescenceimmunoassay (LIA), immunoprecipitation (IP), single radialimmunodiffusion (SRID), turbidimetric immunoassay (TIA), western blot,immunohistochemical staining and the like. Further, examples of aquantification method include ELISA, RIA and the like. Quantificationmay also be performed by a gene analysis method using an array (e.g.,DNA array, protein array). DNA arrays are outlined extensively in (Ed.by Shujunsha, Saibo Kogaku Bessatsu “DNA Maikuroarei to Saishin PCR ho”[Cellular engineering, Extra issue, “DNA Microarrays and Latest PCRMethods”]. Protein arrays are discussed in detail in Nat Genet. 2002December; 32 Suppl: 526-32. Examples of a method of analyzing geneexpression include, but are not limited to, RT-PCR, RACE, SSCP,immunoprecipitation, two-hybrid system, in vitro translation and thelike, in addition to the methods discussed above. Such additionalanalysis methods are described in, for example, Genomu Kaiseki JikkenhoNakamura Yusuke Labo Manyuaru [Genome analysis experimental methodYusuke Nakamura Lab Manual], Ed. by Yusuke Nakamura, Yodosha (2002) andthe like. The entirety of the descriptions therein is incorporatedherein by reference.

As used herein, “amount of expression” refers to the amount ofpolypeptide, mRNA or the like expressed in a cell, tissue or the like ofinterest. Examples of such an amount of expression include amount ofexpression of the polypeptide of the present invention at a proteinlevel assessed by any suitable method including an immunologicalmeasurement method such as ELISA, RIA, fluorescent antibody method,western blot, and immunohistochemical staining by using the antibody ofthe present invention, and the amount of expression of the polypeptideused in the present invention at an mRNA level assessed by any suitablemethod including a molecular biological measuring method such asnorthern blot, dot blot, and PCR. “Change in amount of expression”refers to an increase or decrease in the amount of expression of thepolypeptide used in the present invention at a protein level or mRNAlevel assessed by any suitable method including the above-describedimmunological measuring method or molecular biological measuring method.A variety of detection or diagnosis based on a marker can be performedby measuring the amount of expression of a certain marker.

As used herein, “decrease” or “suppression” of activity or expressionproduct (e.g., protein, transcript (RNA or the like)) or synonymsthereof refers to: a decrease in the amount, quality or effect of aspecific activity, transcript or protein; or activity that decreases thesame. Among decrease, “elimination” refers to activity, expressionproduct or the like being less than the detection limit and especiallyreferred to as “elimination”. As used herein, “elimination” isencompassed by “decrease” or “suppression”.

As used herein, “increase” or “activation” of activity or expressionproduct (e.g., protein, transcript (RNA or the like)) or synonymsthereof refers to: an increase in the amount, quality or effect of aspecific activity, transcript or protein; or activity that increases thesame.

As used herein, “(nucleic acid) primer” refers to a substance requiredfor initiating a reaction of a polymeric compound to be synthesized in apolymer synthesizing enzyme reaction. A synthetic reaction of a nucleicacid molecule can use a nucleic acid molecule (e.g., DNA, RNA or thelike) complementary to a portion of a sequence of a polymeric compoundto be synthesized. A primer can be used herein as a marker detectingmeans.

As used herein, “probe” refers to a substance that can be means forsearch, which is used in a biological experiment such as in vitro and/orin vivo screening. Examples thereof include, but are not limited to, anucleic acid molecule comprising a specific base sequence, a peptidecomprising a specific amino acid sequence, a specific antibody, afragment thereof and the like. As used herein, a probe is used as meansfor marker detection, inspection, or diagnosis.

As used herein, “label” refers to an entity (e.g., substance, energy,electromagnetic wave or the like) for distinguishing a molecule orsubstance of interest from others. Such a method of labeling includes RI(radioisotope) method, fluorescence method, biotin method,chemiluminescent method and the like. When a plurality of markers of thepresent invention or agents or means for capturing the same are labeledby a fluorescence method, labeling is performed with fluorescentsubstances having different fluorescent emission maximum wavelengths. Itis preferable that the difference in fluorescent emission maximumwavelengths is 10 nm or greater. When labeling a ligand, any label thatdoes not affect the function can be used. However, Alexa™ Fluor isdesirable as a fluorescent substance. Alexa™ Fluor is a water-solublefluorescent dye obtained by modifying coumarin, rhodamine, fluorescein,cyanine or the like. This is a series compatible with a wide range offluorescence wavelengths.

Relative to other fluorescent dyes for the corresponding wavelength,Alexa™ Fluor is very stable, bright and has a low level of pHsensitivity. Combinations of fluorescent dyes with fluorescence maximumwavelength of 10 nm or greater include a combination of Alexa™ 555 andAlexa™ 633, combination of Alexa™ 488 and Alexa™ 555 and the like. Whena nucleic acid is labeled, any label can be used that can bind to a baseportion thereof. However, it is preferable to use a cyanine dye (e.g.,Cy3, Cy5 or the like of the CyDye™ series), rhodamine 6G reagent,2-acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) or thelike. Examples of a fluorescent substance with a difference influorescent emission maximum wavelengths of 10 nm or greater include acombination of Cy5 and a rhodamine 6G reagent, a combination of Cy3 andfluorescein, a combination of a rhodamine 6G reagent and fluorescein andthe like. The present invention can utilize such a label to alter asubject of interest to be detectable by the detecting means to be used.Such alteration is known in the art. Those skilled in the art canappropriately carry out such a method in accordance with the label andsubject of interest.

As used herein, “tag” refers to a substance for distinguishing amolecule by a specific recognition mechanism such as receptor-ligand, ormore specifically, a substance serving the role of a binding partner forbinding a specific substance (e.g., having a relationship such asbiotin-avidin or biotin-streptavidin). A tag can be encompassed in thescope of “label”. Accordingly, a specific substance to which a tag isbound can distinguish the specific substance by a contact with asubstrate, to which a binding partner of a tag sequence is bound. Such atag or label is well known in the art. Typical tag sequences include,but are not limited to, myc tag, His tag, HA, Avi Tag™ (Avidity LLC,Aurora, Colo.) and the like. Such a tag may be bound to the marker ofthe present invention or a detection agent, inspection agent, ordiagnostic agent (may be a primer, probe or the like) of the marker.

As used herein, “in vivo” refers to inside of a living body. In specificcontext, “in a living body” refers to the position where a substance ofinterest should be disposed.

As used herein, “in vitro” refers to a state where a portion of a livingbody is extracted or freed “outside of a living body” (e.g., in a testtube) for various research purposes. This is a term that is an antonymof in vivo.

As used herein, when a procedure is performed outside of the body, butthe subject of the procedure is intended to be subsequently returned inthe body, the series of operations is referred to as “ex vivo”. Anembodiment that treats a cell in a living body with an agent of thepresent invention and returns the cell in a patient is also anticipatedin the present invention.

As used herein, “kit” refers to a unit generally providing portions tobe provided (e.g., inspection drug, diagnostic drug, therapeutic drug,antibody, label, manual and the like) into two or more separatesections. This form of a kit is preferred when a composition that shouldnot be provided in a mixed state and is preferably mixed immediatelybefore use for safety or the like is intended to be provided. Such a kitadvantageously comprises an instruction or manual describing how theprovided portions (e.g., inspection drug, diagnostic drug, ortherapeutic drug) are used or how a reagent should be handled. When thekit is used herein as a reagent kit, the kit generally comprises aninstruction describing how to use an inspection drug, diagnostic drug,therapeutic drug, antibody and the like.

As used herein, “instruction” is a document with an explanation of themethod of use of the present invention for a physician or other users.The instruction has a description of the detection method of the presentinvention, method of use of a diagnostic agent, or administration of amedicament or the like. Further, an instruction may have a descriptioninstructing oral administration or administration to the esophagus(e.g., by injection or the like) as a site of administration. Theinstruction is prepared in accordance with a format defined by theregulatory agency of the country in which the present invention ispracticed (e.g., the Ministry of Health, Labor and Welfare in Japan,Food and Drug Administration (FDA) in the U.S. or the like), with anexplicit description showing approval by the regulatory agency. Theinstruction is a so-called package insert and is typically provided in,but not limited to, paper media. The instructions may also be providedin a form such as electronic media (e.g., web sites provided on theInternet or emails).

PREFERRED EMBODIMENTS

Preferred embodiments of the present invention are describedhereinafter. The embodiments are provided hereinafter for betterunderstanding of the present invention. It is understood that the scopeof the present invention should not be limited to the followingdescriptions. Thus, it is apparent that those skilled in the art canreadily make modifications within the scope of the present inventionwhile referring to the descriptions herein. It is understood that thefollowing embodiments of the present invention can be used alone or incombine.

(Therapy and Prevention of Malignant Tumor)

In one aspect, the present invention provides a novel therapeutic orprophylactic drug for malignant tumor. The therapeutic or prophylacticdrug is a therapeutic or prophylactic drug for malignant tumor,comprising an LSR suppressant (e.g., anti-LSR antibody). LSR positivemalignant tumor can be treated or prevented by using such a therapeuticor prophylactic agent. Since such a therapeutic or prophylactic druguses antibodies, it is an excellent drug from the viewpoint of safety.

There are LSR positive and LSR non-positive patients or patients who maybe in such a group in malignant tumor patients or patients who may be insuch a group. For this reason, it is preferable that the therapeuticdrug of the present invention is administered to a patient among themalignant tumor patients who is determined to have malignant tumor thatis LSR positive malignant tumor. In this manner, a drug can beadministered more optimally by diagnosing in advance the presence of LSRpositive.

In one specific embodiment, the composition or medicament (therapeuticdrug, prophylactic drug or the like) of the present invention isformulated in anticipation of implementation in administration to apatient determined to have an episode of LSR positive malignant tumor.

In one embodiment, the LSR suppressant used in the present invention isan antibody, a fragment or a functional equivalent thereof, or a nucleicacid.

In a specific embodiment, the LSR suppressant used in the presentinvention preferably has the ability to inhibit exacerbation due toVLDL. In a more specific embodiment, the LSR antibody of the presentinvention is an antibody having the ability to inhibit exacerbation dueto VLDL. Although not wishing to be bound by any theory, LSRs affectcancer growth by incorporating VLDL and enhancing lipid metabolism. Thatis, it is understood that suppression of LSR functions in cancer cellsexpressing LSRs results in enhanced suppression of cancer cell growth.The antibodies of the present invention can suppress VLDL incorporationinto LSRs of cancer cells to enhance suppression of cancer cell growth.Thus, target cancer or cancer cells of the present invention may becancer or cells associated with VLDL (e.g., cancer shown in the Examplesor cancer associated with cancer cells, ovarian cancer or the like). Ina specific embodiment, the LSR suppressant used in the present inventionis a nucleic acid, which is an antisense nucleic acid, siRNA or thelike. Specifically, the siRNA may comprise SEQ ID NO: 9-14 or the like.

In another embodiment, the LSR suppressant is an antibody or a fragmentor a functional equivalent thereof. The antibody of the presentinvention may be a specific sequence described in other parts of thepresent specification. The antibody may be an antibody or antigenbinding fragment thereof comprising any sequence comprising CDRs of thefull length sequence, or an antibody or antigen binding fragment thereofcomprising a variable region of the following sequence, the frameworkregion thereof comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20or more substitutions, additions, or deletions. The antibody can bemanufactured by using an embodiment described in other parts of thespecification and/or an approach known in the art. For therapy orprevention of the present invention, it is preferable that such anantibody or a fragment or functional equivalent thereof has activity tosuppress LSRs or downstream information transmitting pathway thereof.Such activity may be confirmed by observing the amount of expression oractivity of LSRs, or by directly using malignant tumor cell strains suchas ovarian clear cell adenocarcinoma to observe inhibition of cellgrowth, cytotoxic activity with antibody-dependent cell-mediatedcytotoxicity (ADCC), tumor regression after transplantation into modelanimals or the like. Such approaches are well known in the art, whilethe approach used herein may also be used.

In another aspect, the present invention provides a method of preventingor treating malignant tumor of a subject, comprising administering aneffective amount of LSR suppressant to the subject in need thereof. Itis understood that any form described in other parts of the presentspecification can be used as the LSR suppressant used in theprophylactic or therapeutic method of the present invention.

In another aspect, the present invention also provides a composition ora medicament (therapeutic drug or prophylactic drug) for preventing ortreating malignant tumor, comprising an LSR binding agent. In apreferred embodiment, the composition or medicament (therapeutic drug,prophylactic drug or the like) further comprises a cell-killing agent.Thus, such a composition or medicament (therapeutic drug, prophylacticdrug or the like) may include a complex molecule.

In a specific embodiment, the LSR binding agent is an antibody, afragment or a functional equivalent thereof, or a nucleic acid. In apreferred embodiment, the LSR binding agent is an antibody or a fragmentor a functional equivalent thereof, further bound to a cell-killingagent.

As used herein, “cell-killing agent” is an agent that may dissolve acell membrane. When the agent is a peptide, the cell killing agent iscalled a cytotoxic peptide. Cytotoxic peptide has various nomenclaturesin the art. For example, “soluble peptidic component” and “cell-killingsequence” are also called “cytolic peptide (sequence)”, “cell dissolvingpeptide (sequence)” or the like. However, they are used synonymously inthe content of the present invention. Representative examples of such acytotoxic agent include those listed in Gail D. et al., Cancer Res 2008;68: 9280-9290.; Ian Krop and Eric P. Winer, Clin Cancer Res; 20 (1);1-6. and K Naito et al., Leukemia (2000) 14, 1436-144, as well as, butnot limited to, maytansinoid, emtansine, N-acetyl-γ calicheamicindimethyl hydrazide (NAc-γ calicheamicin, DMH) comprised in CMA-676 andthe like. Representative cell killing peptide includes, but is notlimited to, cell membrane dissolving peptide, cell membrane potentialdestabilizing peptide, cell membrane dissolving/nucleic acid bindingpeptide, and mitochondrial membrane disintegrating peptide.

Such a cell-killing agent may be bound to the binding agent of thepresent invention such as an antibody with a spacer as needed. As usedherein, “spacer” refers to a moiety that forms a chemical bond betweenmolecules of chain-like polymers so as to bridge the molecules. Such aspacer is also called a linker. Representative spacers of a peptideinclude, but are not limited to, a sequence of 0-5 amino acidsconsisting of G and P. A spacer is not essential and may not be present.

A combination of the binding agent of the present invention andcell-killing agent can also be considered a complex molecule. An exampleis provided to explain such a molecule. Such a molecule can be explainedas a molecule made by combining a cytotoxic portion corresponding to theexplosive charge portion and a portion responsible for specificity to acancer cell corresponding to the warhead portion (e.g., peptide/sequencethat specifically binds to a receptor which is highly expressed incancer cells, typically an antibody). When a spacer is used, themolecule would be comprised of a cancer cell specific bindingagent+spacer+cell-killing agent. Any cancer specific binding agent, anyspacer, and any cell-killing agent can be combined herein in any manner.Examples of a manufacturing method and usage method thereof aredescribed. Such a molecule is generally made by a chemical synthesismethod, but when such a molecule is comprised of peptides, a method offorced expression and purification by genetic engineering or a methodcombining such a method can also be used.

For use of the present invention, LSR expression on the cell surface andsensitivity to damage of cancer cells to cell-killing agent areinvestigated for cancer cells to be subjected to therapy. Warhead andexplosive charge are selected base on the result thereof to design anoptimal molecule for the cancer cell. A custom-made peptide toxinobtained from chemical synthesis or the like can be combined as neededwith DDS such as atelocollagen and administered locally or systemicallyfor therapy.

In one embodiment, an LSR binding agent is an antibody or a fragment ora functional equivalent thereof. The antibody can be a sequencespecifically listed in other parts of the present specification.

It is preferable that an administration pathway of a therapeutic drugthat is effective in therapy is used. For example, the administrationpathway may be intravenous, subcutaneous, intramuscular,intraperitoneal, oral administration or the like. The mode ofadministration may be, for example, injection, capsule, tablet, granuleor the like. When an antibody or polynucleotide is administered, usethereof as an injection is effective. An aqueous solution for injectionmay be stored, for example, in a vial or a stainless streel container.Further, an aqueous solution for injection may contain, for example,saline, saccharide (e.g., trehalose), NaCl, NaOH or the like. Further, atherapeutic drug may contain a buffer (e.g., phosphate buffer),stabilizer or the like.

The composition, medicament, therapeutic agent, prophylactic agent andthe like of the present invention generally comprise a therapeuticallyeffective amount of therapeutic agent or effective ingredient and apharmaceutically acceptable carrier or excipient. As used herein,“pharmaceutically acceptable” means that government regulatoryagency-approved or pharmacopoeia or other commonly recognizedpharmacopoeia-listed for use in animals and more specifically in humans.As used herein “carrier” refers to a diluent, adjuvant, excipient orvehicle administered in conjunction with a therapeutic agent. Such acarrier can be an aseptic liquid such as water or oil, including but notlimited to liquids derived from petroleum, animal, plant or synthesis,as well as peanut oil, soybean oil, mineral oil, sesame oil and thelike. When a medicament is orally administered, water is a preferredcarrier. For intravenous administration of a pharmaceutical composition,saline and aqueous dextrose are preferred carriers. Preferably, aqueoussaline solution and aqueous dextrose and glycerol solution are used as aliquid carrier of an injectable solution. Suitable excipients includelight anhydrous silicic acid, crystalline cellulose, mannitol, starch,glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk,silica gel, sodium stearate, glyceryl monostearate, talc, sodiumchloride, powdered skim milk, glycerol, propylene, glycol, water,ethanol, carmellose calcium, carmellose sodium, hydroxypropyl cellulose,hydroxypropyl methylcellulose, polyvinyl acetal diethylamino acetate,polyvinylpyrrolidone, gelatin, medium-chain fatty acid triglyceride,polyoxyethylene hydrogenated castor oil 60, saccharose,carboxymethylcellulose, corn starch, inorganic salt and the like. Whendesired, the composition can contain a small amount of wetting agent oremulsifier or pH buffer. These compositions can be in a form ofsolution, suspension, emulsion, tablet, pill, capsule, powder, sustainedrelease mixture or the like. It is also possible to use traditionalbinding agents and carriers, such as tryglyceride, to prepare acomposition as a suppository. Oral preparation can also comprise astandard carrier such as medicine grade mannitol, lactose, starch,magnesium stearate, sodium saccharin, cellulose, or magnesium carbonate.Examples of a suitable carrier are described in E. W. Martin,Remington's Pharmaceutical Sciences (Mark Publishing Company, Easton, U.S. A). Such a composition contains a therapeutically effective amount oftherapy agent and preferably in a purified form, together with asuitable amount of carrier, such that the composition is provided in aform suitable for administration to a patient. A preparation must besuitable for the administration format. In addition, the composition maycomprise, for example, a surfactant, excipient, coloring agent,flavoring agent, preservative, stabilizer, buffer, suspension,isotonizing agent, binding agent, disintegrant, lubricant, fluidityimproving agent, corrigent or the like.

When the present invention is administered as a medicament, variousdelivery systems are known, and such systems can be used to administer atherapeutic agent of the present invention to a suitable site (e.g.,esophagus). Such a system, for example, can use a recombinant cell thatcan express encapsulated therapeutic agent (e.g., polypeptide) inliposomes, microparticles and microcapsules or use of endocytosismediated by a receptor; construction of a therapy nucleic acid as a partof a retrovirus vector or other vector or the like. The method ofintroduction includes, but not limited to, intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural andoral pathways. A medicament can be administered by any suitable pathway,such as by injection, bolus injection, or by absorption throughepithelial or mucocutaneous lining (e.g., oral cavity, rectum,intestinal mucosa or the like). In addition, an inhaler or mistifierusing an aerosolizing agent can be used as needed. Moreover, otherbiological activating agents can also be administered together.Administration can be systemic or local. When the present invention isused in an ovarian region, a medicament may be administered through anysuitable pathway such as direct injection into an affected site of anovary or the like.

In a specific embodiment where a therapeutic agent is a nucleic acid,the nucleic acid can be constructed as a part of a suitable nucleic acidexpression vector and administered in vivo to be present in a cell topromote expression of an encoded protein. This can be implemented, forexample, by using a retrovirus vector, direct injection, use of amicroparticle gun, coating the nucleic acid with lipid, cell surfacereceptor or transfection agent, or administering a nucleic acid linkedto a tag sequence known to enter the nucleus. Alternatively, a nucleicacid therapeutic agent can be introduced in a cell such that it isincorporated into a host cell DNA by homologous recombination forexpression.

In a preferred embodiment, a composition can be prepared as apharmaceutical composition adapted to administration to humans inaccordance with a known method. Such a composition can be administeredby an injection. A composition for injection is typically a solution inan aseptic isotonic aqueous buffer. A composition can also comprise alocal anesthetic such as lidocaine which alleviates the pain at the siteof injection and a solubilizing agent as needed. Generally, ingredientscan be supplied separately or by mixing the ingredients together in aunit dosing form and supplied, for example, in a sealed container suchas an ampoule or sachet showing the amount of active agent or as alyophilized powder or water-free concentrate. When a composition is tobe administered by injection, the composition can be distributed byusing an injection bottle containing aseptic agent-grade water orsaline. When a composition is to be administered by injection, anaseptic water or saline ampoule for injection can also be provided suchthat the ingredients can be mixed prior to administration.

The composition, medicament, therapeutic agent, and prophylactic agentof the present invention can be prepared as a neutral or salt form orother prodrugs (e.g., ester or the like). Pharmaceutically acceptablesalts include salts formed with a free carboxyl group, derived fromhydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaricacid or the like, salts formed with a free amine group, derived fromisopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaineor the like, and salts derived from sodium, potassium, ammonium,calcium, or ferric hydroxide or the like.

The amount of therapeutic agent of the present invention that iseffective in therapy of a specific disorder or condition may varydepending on the properties of the disorder or condition. However, suchan amount can be determined by those skilled in the art by a standardclinical technique based on the descriptions herein. Furthermore, an invitro assay can be used in some cases to assist the identification ofthe optimal dosing range. The precise dose to be used in a preparationmay also vary depending on the administration pathway or the severity ofthe disease or disorder. Thus, the dose should be determined inaccordance with the judgment of the attending physician or the conditionof each patient. The dosage is not particularly limited, but may be0.001, 1, 5, 10, 15, 100 or 1000 mg/kg body weight per dosage or withina range between any two values described above. The dosing interval isnot particularly limited, but may be, for example, 1 or 2 administrationevery 1, 7, 14, 21, or 28 days or 1 or 2 administrations in the range ofperiod between any two values described above. The dosage, dosinginterval, and dosing method may be appropriately selected depending onthe age, weight, symptom, target organ or the like of the patient.Further, it is preferable that a therapeutic drug contains atherapeutically effective amount, or an amount effective for exerting adesired effect, of effective ingredients. When a malignant tumor markersignificantly decreases after administration, the presence of atherapeutic effect may be acknowledged.

“Patient” in one embodiment of the present invention includes humans andmammals excluding humans (e.g., one or more types of mice, guinea pigs,hamsters, rats, rabbits, pigs, sheep, goats, cows, horses, cats, dogs,marmosets, monkeys and the like). Further, the patient may be a patientdetermined or diagnosed as having an episode of LSR positive malignanttumor. It is preferable that determination or diagnosis in this regardis performed by detecting the LSR protein level.

The pharmaceutical composition, therapeutic agent or prophylactic agentof the present invention can be provided as a kit.

In a specific embodiment, the present invention provides an agent packor kit comprising one or more containers filled with one or moreingredients of the composition or medicament of the present invention.Optionally, information indicating approval for manufacture, use or salefor administration to a human by a government agency regulating themanufacture, use or sale of medicaments or biological products in astipulated form can be appended to such a container.

The kit of the present invention can also contain an expression vectorencoding a protein to be used as the composition, therapeutic agent,prophylactic agent or medicament of the present invention. Since such aprotein, after expression, forms a biologically active complex, theprotein may be reconstituted. Such a kit preferably contains a requiredbuffer and a reagent. Optionally, instruction (package insert) for useof the kit and/or information indicating approval for manufacture, useor sale for administration to a human by a government agency regulatingthe manufacture, use or sale of medicaments or biological products in astipulated form can be appended to such a container.

In a specific embodiment, the pharmaceutical composition comprising anucleic acid of the present invention can be administered via liposomes,microparticles, or microcapsules. In various embodiments of the presentinvention, it may be useful to use such a composition to achievesustained release of the nucleic acid.

One embodiment of the present invention may be an anti-LSR antibody,which is one or more antibodies selected from the group consisting of(a) an antibody comprising heavy chain CDRs 1, 2, and 3 and light chainCDRs 1, 2, and 3 with amino acid sequences set forth in positions 31-35,50-66, 99-104, 153-165, 182-188 and 221-230 of SEQ ID NO: 1,respectively, (b) an antibody comprising heavy chain CDRs 1, 2, and 3and light chain CDRs 1, 2, and 3 with amino acid sequences set forth inpositions 31-35, 50-66, 99-103, 152-165, 182-188 and 221-230 of SEQ IDNO: 2, respectively, (c) an antibody comprising heavy chain CDRs 1, 2,and 3 and light chain CDRs 1, 2, and 3 with amino acid sequences setforth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and 221-229 ofSEQ ID NO: 3, respectively, (d) an antibody comprising heavy chain CDRs1, 2, and 3 and light chain CDRs 1, 2, and 3 with amino acid sequencesset forth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and221-229 of SEQ ID NO: 4, respectively, (e) an antibody comprising heavychain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 with amino acidsequences set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188and 221-229 of SEQ ID NO: 5, respectively, and (f) an antibodycomprising heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3with amino acid sequences set forth in positions 31-35, 50-66, 99-104,153-165, 182-188 and 221-229 of SEQ ID NO: 6, respectively, or a mutantof the antibody, which is free of a mutation in the CDRs but comprisesone or several substitutions, additions, or deletions in a framework ofthe antibody in the mutant. Use of such an anti-LSR antibody caneffectively suppress especially the growth of LSR-positive malignanttumor cells. Further, LSR-positive malignant tumor can be efficientlydiagnosed. Further, another embodiment of the present invention is ananti-LSR antibody comprising at least one of the sets of amino acidsequences of heavy chain CDRs 1, 2, and 3 listed above. These antibodiesmay be an antibody selected from a monoclonal antibody, polyclonalantibody, chimeric antibody, humanized antibody, human antibody,multifunctional antibody, bispecific or oligospecific antibody, singlechain antibody, scFV, diabody, sc(Fv)₂ (single chain (Fv)₂), andscFv-Fc.

The anti-LSR antibody according to one embodiment of the presentinvention may comprise a set of amino acid sequences of heavy chain CDRs1, 2, and 3 and light chain CDRs 1, 2, and 3, and at least one,preferably, 2, 3, 4, 5, 6, 7 or all of the heavy chain FRs 1, 2, 3, and4 and light chain FRs 1, 2, 3, and 4 are identical, substantiallyidentical, or identical except for a conservative substitution with anyone of SEQ ID NOs: 1-6. The anti-LSR antibody may be one or more typesof antibodies. Further, another embodiment of the present invention isan anti-LSR antibody comprising at least one of the amino acid sequenceset of heavy chain FRs 1, 2, 3, and 4 listed above.

The anti-LSR antibody according to one embodiment of the presentinvention may be in a form of scFv. In such a case, a linker between aheavy chain and a light chain may have an amino acid sequence set forthin positions 116-132 of SEQ ID NO: 1, positions 116-132 of SEQ ID NO: 2,positions 116-132 of SEQ ID NO: 3, positions 116-132 of SEQ ID NO: 4,positions 116-132 of SEQ ID NO: 5, or positions 116-132 of SEQ ID NO: 6.

VHs of #9-7, #16-6, No. 26-2, No. 27-6, No. 1-25, and No. 1-43 describedin Example 2 described below are positions 1-115 of SEQ ID NO: 1,positions 1-115 of SEQ ID NO: 2, positions 1-115 of SEQ ID NO: 3,positions 1-115 of SEQ ID NO: 4, positions 1-115 of SEQ ID NO: 5, andpositions 1-115 of SEQ ID NO: 6, respectively. Further, VLs of #9-7,#16-6, No. 26-2, No. 27-6, No. 1-25, and No. 1-43 described in Example 2described below are positions 133-238 of SEQ ID NO: 1, positions 133-239of SEQ ID NO: 2, positions 133-238 of SEQ ID NO: 3, positions 133-238 ofSEQ ID NO: 4, positions 133-238 of SEQ ID NO: 5, and positions 133-238of SEQ ID NO: 6, respectively.

The amino acid sequences listed above may be one or more amino acidsequences selected from the group consisting of (i) the above-describedamino acid sequence with one or several base sequence deletions,substitutions, insertions or additions, (ii) an amino acid sequence with90% or greater homology to the above-described amino acid sequence, and(iii) an amino acid sequence encoded by a polynucleotide that hybridizesspecifically to a polynucleotide consisting of a base sequencecomplementary to a base sequence encoding the above-described amino acidunder stringent conditions, as long as an anti-LSR antibody has adesired effect.

A vector or polynucleotide encoding the anti-LSR antibody according toone embodiment of the present invention can be introduced into a cell toproduce a transformant. Such a transformant can be used to make theanti-LSR antibody according to the embodiment of the present invention.The transformant may be a cell of a human or a mammal excluding humans(e.g., rat, mouse, guinea pig, rabbit, cow, monkey or the like).Examples of a mammalian cell include Chinese hamster ovary cells (CHOcells), monkey cells COS-7 and the like. Further, the tranformant may beEscherichia bacteria, yeasts or the like.

For example, an E. coli derived plasmid (e.g., pET-Blue), a Bacillussubtilis derived plasmid (e.g., pUB110), a yeast derived plasmid (e.g.pSH19), an animal cell expression plasmid (e.g., pA1-11,pdDNA3.1-V5/His-TOPO), bacteriophage such as A phage, a virus-derivedvector or the like can be used as the above-described vector. Suchvectors may comprise a constituent element required for proteinexpression such as a promoter, origin of replication, or antibioticresistant gene. The vector may be an expression gene.

Examples of method of introducing the above-described polynucleotide orvector into cells that can be used include calcium phosphate method,lipofection, electroporation, method using adenovirus, method using aretrovirus, and microinjection (Revised 4th edition Shin IdenshikogakuHandobukku [New Genetic Engineering Handbook], Yodosha (2003): 152-179).Examples of a method of producing an antibody using a cell that can beused include the methods described in “Tanpakushitsu Jikken Handobukku[Protein experiment handbook], Yodosha (2003): 128-142”. Purification ofantibodies can use, for example, ammonium sulfate, ethanolprecipitation, protein A, protein G, gel filtration chromatography,anion, cation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxyapatite chromatography, lectin chromatography or the like“Tanpakushitsu Jikken Handobukku [Protein experiment handbook], Yodosha(2003): 27-52”.

To implement the present invention, a nucleic acid can be selected asthe suppressant in a nucleic acid form of the present invention by usingantisense activity as an indicator. In this regard, “antisense activity”refers to activity that can specifically suppress or decrease expressionof a target gene. More specifically, antisense activity refers toactivity that can decrease the amount of protein expression, dependingon the nucleotide sequence introduced into cells, by specificallyreducing the amount of mRNA of a gene having a nucleotide sequenceregion complementary to such a sequence. The approach thereof is roughlyclassified into a method of introducing an RNA molecule complementary tomRNA made from a target gene directly into cells, and a method ofintroducing a construct vector that can express an RNA complementary toa gene of interest into cells.

Antisense activity is achieved by a nucleic acid sequence with a lengthof at least 8 contiguous nucleotides, which is complementary to anucleic acid sequence of a gene of interest. Such a nucleic acidsequence may be a nucleic acid sequence preferably with a length of atleast 9 contiguous nucleotides, more preferably with a length of 10contiguous nucleotides, and still more preferably with a length of 11contiguous nucleotides, a length of 12 contiguous nucleotides, a lengthof 13 contiguous nucleotides, a length of 14 contiguous nucleotides, alength of 15 contiguous nucleotides, a length of 16 contiguousnucleotides, a length of 17 contiguous nucleotides, a length of 18contiguous nucleotides, a length of 19 contiguous nucleotides, a lengthof 20 contiguous nucleotides, a length of 21 contiguous nucleotides, alength of 22 contiguous nucleotides, a length of 23 contiguousnucleotides, a length of 24 contiguous nucleotides, a length of 25contiguous nucleotides, a length of 30 contiguous nucleotides, a lengthof 40 contiguous nucleotides, or a length of 50 contiguous nucleotides.Such a nucleic acid sequence includes nucleic acid sequences that are atleast 70% homologous, more preferably at least 80% homologous, stillmore preferably 90% homologous or 95% homologous to the aforementionedsequences. Such antisense activity is preferably complementary to asequence at the 5′ terminus of a nucleic acid sequence of a gene ofinterest. Such an antisense nucleic acid sequence includes theaforementioned sequences with one or several or one or more nucleotidesubstitutions, additions, and/or deletions. Thus, antisense activity asused herein includes, but is not limited to, decrease in the amount ofgene expression.

Common antisense techniques are described in text books (Murray, J A Heds., Antisense RNA and DNA, Wiley-Liss Inc, 1992). Furthermore, thelatest research has elucidated a phenomenon called RNA interference(RNAi), leading to development of antisense techniques.

As used herein, “RNAi” is an abbreviation of “RNA interference” and iscommonly known in the art. RNA interference is a biological process thatinhibits or downregulates gene expression in cells, mediated by an agentinducing RNAi. For example, RNA interference refers to a phenomenon ofspecific degradation of homologous mRNA to suppress the synthesis ofgene products by introducing into a cell an agent inducing RNAi, such asa double stranded RNA (also called dsRNA), or a technique used therein.As used herein, “RNAi” may in some cases be used synonymously with“agent inducing RNAi”, “agent causing RNAi”, “RNAi agent” or the like.For RNAi, see, for example, Zamore and Haley, 2005, Science, 309,1519-1524; Vaughn and Martienssen, 2005, Science 309, 1525-1526; Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al,International Publication No. WO 00/44895; Zernicka-Goetz et al,International Publication No. WO 01/36646; Fire, InternationalPublication No. WO 99/32619; Plaetinck, et al., InternationalPublication No. WO 00/01846; Mello and Fire, International PublicationNo. WO 01/29058; Deschamps-Depaillette, International Publication No. WO99/07409 and Li et al., International Publication No. WO 00/44914;Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237; Hutvagner and Zamore, 2002, Science,297, 2056-60; McManus et al., 2002, RNA, 8, 842-850; Reinhart et al.,2002, Gene & Dev., 16, 1616-1626; and Reinhart & Bartel, 2002, Science,297, 1831. Further, it is understood that the term RNAi as used hereinrepresents a synonym of other terms used to describe sequence specificRNA interference such as post-transcription gene silencing, inhibitionof translation, inhibition of transcription, or epigenetics. As usedherein, “agent causing RNAi” may be any agent as long as “RNAi” iscaused.

Examples of “agent causing RNAi” as used herein include “smallinterfering nucleic acid” “siNA”, “small interfering RNA”, “siRNA”,“small interfering nucleic acid molecule”, “small oligonucleotidemolecule”, “chemically modified small interfering nucleic acid molecule”and the like. These terms refer to any nucleic acid molecule that caninhibit or downregulate gene expression or virus replication by sequencespecifically mediating RNA interference “RNAi” or gene silencing. Theseterms may represent an individual nucleic acid molecule, multiple suchnucleic acid molecules, or a pool of such nucleic acid molecules. Themolecules may be a double stranded nucleic acid molecule comprising aself-complementary sense region and an antisense region.

“SiRNA” that is typically used in the present invention is a doubledstranded RNA that is short with a length of generally about 20 bases(e.g., typically about 21-23 bases long) or less. Such an siRNA, whenexpressed in cells, suppresses gene expression and suppresses expressionof a target pathogenic gene of the siRNA. Thus, such an siRNA can beused in therapy, prevention, prognosis or the like of a disease. ThesiRNA used in the present invention may be in any form, as long as it iscapable of inducing RNAi.

In the present invention, an antisense region in an agent causing RNAisuch as an siRNA comprises a sense region having a nucleotide sequencewhich is complementary to a nucleotide sequence in a target nucleic acidmolecule or a portion thereof and a nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof. These moleculescan be assembled from two separate oligonucleotides, one being a sensestrand and the other being an antisense strand. The antisense strand andsense strand in this regard are self-complementary (i.e., each strandcomprises a nucleotide sequence that is complementary to the nucleotidesequence in the other strand, e.g., the antisense strand and the sensestrand form a double strand or double stranded structure). A doublestranded region in this regard can be, for example, about 15 to about 30base pairs such as about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29 or 30 base pairs or longer. The antisense strand comprises anucleotide sequence that is complementary to a nucleotide sequence in atarget nucleic acid molecule or a portion thereof, and the sense strandcomprises a nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15-25 or more nucleotides ofthe molecule are complementary to a target nucleic acid or a portionthereof). Alternatively, these molecules are assembled from a singleoligonucleotide, and the self-complementary sense region and antisenseregion of these molecules are linked by a nucleic acid linker or anon-nucleic acid linker. These molecules can be polynucleotides having adouble stranded, asymmetrical double stranded, hairpin, or asymmetricalhairpin secondary structure comprising a self-complementary sense regionand antisense region. The antisense region in this regard comprises aseparate sense region having a nucleotide sequence which iscomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof and a nucleotide sequence corresponding to thetarget nucleic acid sequence or a portion thereof. These molecules maybe a cyclic single stranded polynucleotide having two or more loopstructures and a stem comprising a self-complementary sense region andantisense region. The antisense region in this regard comprises aseparate sense region having a nucleotide sequence which iscomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof and a nucleotide sequence corresponding to thetarget nucleic acid sequence or a portion thereof. In addition, a cyclicpolynucleotide can be processed in vivo or in vitro to generate anactive molecule that can mediate RNAi. These agents may also comprise asingle stranded polynucleotide having a nucleotide sequence, which iscomplementary to a nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for instance, for these agents, a nucleotidesequence corresponding to the target nucleic acid molecule or a portionthereof does not need to be present in these agents). A single strandedpolynucleotide may further comprise a terminal phosphoric acid groupsuch as 5′ phosphoric acid (for example, see Martinez et Al., 2002,Cell., 110, 563-574 and Schwarz et al., 2002, Molecular Cell, 10,537-568) or 5′, 3′-diphosphoric acid. In a certain embodiment, the LSRsuppressant of the present invention comprises separate sense andantisense sequences or regions. The sense region and antisense region inthis regard are covalently attached by a nucleotide or non-nucleotidelinker molecule known in the art, or non-covalently attached to eachother by ionic interaction, hydrogen bond, Van der Waal's interaction,hydrophobic interaction and/or stacking interaction. In a certainembodiment, the LSR suppressant of the present invention comprises anucleotide sequence that is complementary to a nucleotide sequence of atarget gene. In another embodiment, the LSR suppressant of the presentinvention interacts with a nucleotide sequence of a target gene suchthat expression of the target gene is inhibited. The LSR suppressant isnot necessarily limited herein to molecules comprising only an RNA. TheLSR suppressant also encompasses chemically modified nucleotides andnon-nucleotides. In a certain embodiment, when the present invention isa small interfering nucleic acid molecule, a 2′ hydroxy (2′-OH)containing nucleotide may be lacking. In a certain embodiment, thepresent invention can be a small interfering nucleic acid, which doesnot require the presence of a nucleotide having a 2′ hydroxyl group formediating RNAi. Thus, when the present invention is a small interferingnucleic acid molecule, ribonucleotide (e.g., nucleotide having a 2′-OHgroup) does not need to be included. However, when the presence of aribonucleotide in an LSR suppressant is not required for maintainingRNAi, it may have a bound linker, or another bound or conjugated group,moiety or strand comprising one or more nucleotides having a 2′-OHgroup. Optionally, an agent suppressing LSRs of the present inventionmay comprise a ribonucleotide in about 5, 10, 20, 30, 40 or 50% of thenucleotide positions. Herein, the LSR suppressant may be a nucleic acidmolecule that can mediate sequence specific RNAi, such as smallinterfering RNA (siRNA), double stranded RNA (dsRNA), microRNA (miRNA),short hairpin RNA (shRNA), small interfering oligonucleotide, smallinterfering nucleic acid, small interfering modified oligonucleotide,chemically modified siRNA, or post-transcriptional gene silencing RNA.

Examples of agents inducing RNAi herein include, but are not limited to,RNAs comprising a double stranded moiety with a length of at least 10nucleotides, comprising a sequence having at least about 70% homology ora sequence that hybridizes under stringent conditions to a portion of anucleic acid sequence of a target gene and variants thereof. The agentin this regard can preferably comprise a 3′ overhang, and morepreferably the 3′ overhang is a DNA with a length of 2 nucleotides orlonger (e.g., DNA with a length of 2-4 nucleotides).

Alternatively, examples of RNAi used in the present invention include,but are not limited to, a pair of short complementary sequences in theopposite direction (e.g., 15 bp or longer such as 24 bp or the like).

Although not wishing to be bound by any theory, as one conceivableworking mechanism of RNAi, when a molecule inducing RNAi such as dsRNAis introduced into cells for a relatively long (e.g., 40 base pairs orgreater) RNA, an RNase III-like nuclease called a dicer having ahelicase domain cuts out the molecule into about 20 base pair each fromthe 3′ terminus in the presence of ATP to produce short dsRNA (alsocalled siRNA). As used herein, “siRNA” is an abbreviation for shortinterfering RNA and refers to a short double stranded RNA with 10 basepairs or more prepared by artificial chemical synthesis or biochemicalsynthesis, synthesis in the body of an organism, or degradation of adouble stranded RNA with about 40 bases or more in vivo. An siRNAgenerally has a 5′-phosphoric acid or 3′-OH structure, and the 3′terminus overhangs by about 2 bases. A specific protein binds to thesiRNA to form an RISC (RNA-induced-silencing-complex). Such a complexrecognizes and binds to an mRNA having the same sequence as the siRNAand cleaves the mRNA in the middle portion of the siRNA by RNaseIII-like enzymatic activity. The relationship of the siRNA sequence andmRNA sequence to be cleaved as a target is preferably a 100% match.However, for a mutation of a base at a position away from the middle ofthe siRNA, cleaving activity due to RNAi would not be completely lost,but instead partially remains. On the other hand, a mutation of a basein the middle portion of the siRNA has a significant effect, such thatmRNA cleaving activity due to RNAi is dramatically reduced. For mRNAswith a mutation, such a property can be utilized to degrade only mRNAscomprising a specific mutation by synthesizing an siRNA with themutation positioned in the middle and introducing the siRNA into cells.Thus, the present invention can use an siRNA itself as an agent inducingRNAi or an agent that would produce an siRNA (e.g., typically a dsRNAwith about 40 or more bases) as such an agent.

Although not wishing to be bound by any theory, it is intended forsiRNAs that, aside from the above-described pathway, an antisense strandof the siRNA binds to an mRNA and acts as a primer of an RNA-dependentRNA polymerase (RdRP), such that a dsRNA is synthesized and the dsRNA isused again as a substrate of a dicer to produce a new siRNA and amplifythe action. Thus, the siRNA itself and agents producing an siRNA arealso useful in the present invention. In fact, for example, 35 dsRNAmolecules nearly completely degrade 1000 or more mRNA copies in cells ininsects or the like. Thus, it is understood that the siRNA itself andagents producing an siRNA are also useful.

In another embodiment, the agent inducing RNAi of the present inventioncan be a short hairpin structure (shRNA; short hairpin RNA) having anoverhang at the 3′ terminus. As used herein, “shRNA” refers to amolecule with about 20 or more base pairs, which comprises a partiallypalindrome-like base sequence in a single stranded RNA to be in a doublestranded structure in a molecule to have a hairpin-like structure. Suchan shRNA is artificially made by chemical synthesis. Alternatively, suchan shRNA can be produced by synthesizing a hairpin structure DNAcomprising DNA sequences of sense and antisense strands linked inopposite directions in vitro into an RNA with a T7RNA polymerase.Although not wishing to be bound by any theory, it should be understoodthat such an shRNA, after introduction into cells, is degraded into alength of about 20 bases (typically, for example, 21 bases, 22 bases or23 bases) in the cells and induces RNAi as in an siRNA, resulting in atreatment effect of the present invention. It should be understood thatsuch an effect is exerted in a wide range of organisms such as insects,plants and animals (including mammals). Since an shRNA induces RNAi asin siRNAs in this manner, it can be used as an effective ingredient ofthe present invention. Further, an shRNA preferably can have a 3′overhang. The length of a double stranded moiety is not particularlylimited, but the length can be preferably about 10 nucleotides long orlonger and more preferably about 20 nucleotides long or longer. The 3′overhang in this regard can be preferably a DNA, more preferably a DNAwith a length of at least two nucleotides or more, and still morepreferably a DNA with a length of 2-4 nucleotides. The agent inducingRNAi used in the present invention can be artificially synthesized(e.g., chemically or biochemically) or naturally occurring. There is nofundamental difference in the effect of the present inventiontherebetween. A chemically synthesized agent is preferably purified byliquid chromatography or the like.

The agent inducing RNAi used in the present invention can also besynthesized in vitro. In such a synthesis system, a T7RNA polymerase andT7 promoter are used to synthesize antisense and sense RNAs from atemplate DNA. After annealing is performed thereon in vitro, RNAi isinduced through the aforementioned mechanism when cells are introducedto achieve the effect of the present invention. In this regard, such anRNA can be introduced into cells, for example, by any suitable methodsuch as the calcium phosphate method. Examples of the agents inducingRNAi of the present invention include agents such as a single strandthat can hybridize with an mRNA or all similar nucleic acid analogsthereof. Such agents are also useful in the present invention.

One embodiment of the present invention is a therapeutic drug for LSRpositive malignant tumor comprising an RNAi molecule directed to LSRs ora polynucleotide encoding the RNAi molecule. Growth of LSR positivemalignant tumor cells can be suppressed when such an RNAi molecule orpolynucleotide encoding the RNAi molecule is used. “Polynucleotide” inone embodiment of the present invention may be a polymeric compoundhaving 10 or more nucleotides, comprising a nucleotide polymerized in astraight chain.

“RNAi molecule” in one embodiment of the present invention is an RNAstrand having RNAi action. Examples thereof include siRNA, shRNA, miRNA,small RNA having RNAi action and the like.

“RNAi” in one embodiment of the present invention includes a phenomenonof suppressing or silencing a function of a target gene, mRNA or thelike by one or more of siRNA, shRNA, miRNA, single or double strandedRNA with a short or long chain, modified products thereof and the like.

For example, siDirect 2.0 (Naito et al., BMC Bioinformatics. 2009 Nov.30; 10: 392.) or the like can be used to design an RNAi molecule.Further, designing can be commissioned to a specialist company (e.g.,Takara Bio Inc. or the like). RNAi action can be verified byquantification of the amount of RNA strand expression by real-timeRT-PCR. RNAi action can also be confirmed by analysis of the amount ofRNA strand expression by Northern blot or a method of analyzing theamount of protein and observing the phenotype or the like by Westernblot. Further, a plasmid producing siRNAs or shRNAs for a specific genecan be purchased, for example, from a specialist company (e.g., TakaraBio Inc. or the like).

“SiRNA” in one embodiment of the present invention comprises an RNAstrand capable of inducing RNAi. Two strands of an siRNA can generallybe separated into a guide strand and a passenger strand, where the guidestrand in incorporated into an RISC. The guide strand incorporated intothe RISC is used to recognize a target RNA. Although an artificiallycreated guide strand is mainly used in RNAi research, those endogenousin a living body are also known. The above-described guide chain may becomposed of an RNA with 15 bases or more. When there are 15 bases ormore, the possibility of being able to precisely bind to a targetnucleotide increases. Further, the guide strand may be composed of anRNA with 40 bases or less. With 40 bases or less, the risk of adisadvantageous phenomenon such as interferon response occurring isfurther reduced.

“shRNA” in one embodiment of the present invention comprises a singlestrand of RNA strand that can induce RNAi and form a structure foldedinto a hairpin shape (hairpin-like structure). Typically, an shRNA iscleaved by a dicer in a cell to cut out an siRNA. It is known that atarget RNA is cleaved by the siRNA. The above-described shRNA may becomposed of 35 or more nucleotides. With 35 or more, the possibility ofbeing able to precisely form a hairpin-like structure unique to shRNAsincreases. Further, the above-described shRNA may be composed of an RNAwith 100 bases or less. With 100 bases or less, the risk of adisadvantageous phenomenon such as interferon response occurring isreduced. However, many of the pre-miRNAs that generally have a similarstructure and function as shRNAs have a length of about 100 nucleotidesor more. Thus, it is conceivable that they can function as a shRNA evenwhen the length of the shRNA is not necessarily 100 bases or less.

It is known that “miRNA” in one embodiment of the present inventioncomprises an RNA strand having a function similar to an siRNA andsuppresses translation of, and degrades, a target RNA strand. Thedifference in miRNAs and siRNAs is generally in the production pathwayand the detailed mechanism.

“Small RNA” in one embodiment of the present invention refers to arelatively small RNA strand. Examples thereof include siRNAs, shRNAs,miRNAs, antisense RNAs, small RNAs with one or two strands and the like.

The RNAi molecules may comprise an overhang consisting of 1-5 bases atthe 5′ terminus or the 3′ terminus. It is understood that RNAiefficiency is enhanced in such a case. The number of bases may be, forexample, 5, 4, 3, 2, or 1, or within a range of any two values describedabove. When the above-described RNAi molecule is double stranded, amismatching RNAs may be present between each RNA strand. The number ofmismatching RNAs may be, for example, 1, 2, 3, 4, 5, or 10 or less, orwithin the range of any two values described above. Further, theabove-described RNAi molecule may comprise a hairpin loop. The number ofbases of a hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 orwithin any two values described above. The base sequence may have one ora plurality of base sequence deletions, substitutions, insertions oradditions, as long as the sequence has a desired effect. The left sideof each base sequence is denoted as the 5′ terminus and the right sideas the 3′ terminus.

The length of the above-described RNAi molecule may be, for example, 15,18, 20, 25, 30, 40, 50, 60, 80, 100, 200, or 400 bases or within a rangebetween any two values described above. The number is preferably 15 ormore or 100 or less from the viewpoint of improving the therapeuticeffect on LSR positive malignant tumor.

“RNA strand” in one embodiment of the present invention includes thoseconstituted in a form in which a plurality of RNAs or equivalentsthereof are bound. “DNA strand” in one embodiment of the presentinvention includes those constituted in a form in which a plurality ofDNAs or equivalents thereof are bound. The RNA strand or DNA strandincludes RNA strands or DNA strands in a single stranded ormulti-stranded (e.g., double stranded) form. The RNA strand or DNAstrand may be bound to a substance promoting incorporation into cells(e.g., PEG or derivative thereof), labeling tag (e.g., fluorescentlabeling tag or the like), a linker (e.g., nucleotide linker or thelike) or the like. The RNA strand or DNA strand can be synthesized byusing a nucleic acid synthesizer or purchased from a specialist company(e.g., Invitrogen or the like). An RNA strand or DNA strand in a livingbody may form a salt or a solvate. Further, an RNA strand or DNA strandin a living body may be chemically modified. The term RNA strand or DNAstrand includes, for example, RNA strands or DNA strands forming a saltor solvate, RNA strands or DNA strands subjected to chemicalmodification, and the like. Further, an RNA strand or DNA strand may bean analog of the RNA strand or an analog of the DNA strand.

Examples of “salt” in one embodiment of the present invention includeanionic salts formed with any acidic (e.g., carboxyl) group and cationicsalts formed with any basic (e.g., amino) group. Salts include inorganicsalts and organic salts, as well as salts described in, for example,Berge et al., J. Pharm. Sci., 1977, 66, 1-19. Further examples includemetal salts, ammonium salts, salts with organic base, salts withinorganic acid, salts with organic acid and the like. “Solvent” in oneembodiment of the present invention is a compound formed with a soluteor solvent. For example, J. Honig et al., The Van Nostrand Chemist'sDictionary P650 (1953) can be referred for solvates. When a solvent iswater, a solvate formed is a hydrate. It is preferable that the solventdoes not obstruct the biological activity of the solute. Examples ofsuch a preferred solvent include, but not particularly limited to waterand various buffers. Examples of “chemical modification” in oneembodiment of the present invention include modification with PEG or aderivative thereof, fluorescein modification, biotin modification andthe like.

The above-described RNAi molecule preferably comprises a base sequencethat is complementary to a portion of a base sequence of an LSR mRNAfrom the viewpoint of stably exerting RNAi action. The above-described“portion” may be, for example, 5, 10, 15, 18, 20, 22, 24, 26, 28, 30,35, 40 or 50 bases or more or within a range of any two values describedabove.

The siRNA used in Example 3 described below comprises the base sequenceof SEQ ID NO: 9 or 10. These base sequences are considered to be basesequences complementary to a portion of an LSR mRNA and responsible forthe function as a guide strand. One embodiment of the present inventioncomprises an RNAi molecule comprising such the base sequence of SEQ IDNO: 9 or 10. The RNAi molecule may further comprise a base sequencecomplementary to the base sequence set forth in SEQ ID NO: 9 or 10(e.g., SEQ ID NO: 11 or 12, respectively). “Complementary base sequence”in one embodiment of the present invention is abase sequence having apolynucleotide with high complementarity capable of hybridizing toanother polypeptide. The full length sense strand of the siRNA used inExample 3 described below is the base sequence of SEQ ID NO: 13 or 14,and the full length antisense strand is the base sequence of SEQ ID NO:15 or 16.

As long as an LSR siRNA has a desired effect, the base sequences listedabove may be (i) an amino acid sequence with one or several basesequence deletions, substitutions, insertions or additions in theabove-described base acid sequence, or (ii) a base sequence encoded by apolynucleotide that specifically hybridizes with a polynucleotideconsisting of a base sequence complementary to the above-described basesequence under stringent conditions.

One embodiment of the present invention is a therapeutic drug for LSRpositive malignant tumor, comprising an LSR antagonist. The LSRantagonist comprises a substance inhibiting the expression or functionof an LSR. The growth of LSR positive malignant tumor cells can besuppressed by using such an LSR antagonist. The form of antagonist isnot particularly limited as long as it has an action of inhibiting theexpression or function of an LSR. For example, the antagonist may be ina form of an antibody, RNA strand, DNA strand, low molecular weightorganic compound, or polypeptide. The above-described RNA strand may bean RNAi molecule directed to an LSR. A DNA strand encoding an RNAimolecule directed to an LSR can be used as the above-described DNAstrand. For example, the DNA strand may be in a form of a vector.

Examples of “inhibit the expression of a protein” in one embodiment ofthe present invention include inhibiting the transcription mechanismfrom a gene to an mRNA or inhibiting the translation mechanism from anmRNA to a protein. Examples further include inducing degradation of agene, mRNA or protein to ultimately decrease the amount of protein.“Inhibit a function of protein” in one embodiment of the presentinvention includes causing a structural change in a protein to reducethe activity of the protein. Examples thereof further include inhibitingthe expression of a gene, resulting in reduction in the amount of mRNAor protein production.

“State where expression is inhibited” in one embodiment of the presentinvention includes a state of significantly decreased amount ofexpression relative to normal levels. The amount of mRNA or protein maybe used as an indicator for the amount of expression. “Significantly” inone embodiment of the present invention may be, for example, a statewhere there is a statistically significant difference, when assessed byStudent's t-test (one or two tailed), at p<0.05. Further, a state wherea substantial difference has occurred is also included. “State where afunction is inhibited” in one embodiment of the present inventionincludes a state with significantly decreased activity relative tonormal levels.

One embodiment of the present invention is a novel method of therapy formalignant tumor. Such a therapeutic method comprises, for example, astep of administering an anti-LSR antibody to a patient. LSR positivemalignant tumor can be treated by using such a therapeutic method.Further, such a therapeutic method is excellent from the viewpoint ofsafety, as the method uses antibodies.

There are LSR positive and LSR non-positive patients among malignanttumor patients. For this reason, the above-described therapeutic methodis preferably administered to a malignant tumor patient determined tohave malignant tumor that is LSR positive malignant tumor. In thismanner, diagnosis for the presence or absence of an LSR positivecondition in advance enables a more optimal dosing.

Thus, the above-described therapeutic method for malignant tumorpreferably comprises a step of diagnosing whether a patient has anepisode of LSR positive malignant tumor from the viewpoint ofadministering a more optimal dosing. Further, the therapeutic method maycomprise a step of investigating whether malignant tumor cells derivedfrom a patient express LSRs. An episode of LSR positive malignant tumormay be diagnosed, for example, by diagnosing mRNA expression or proteinexpression. The diagnosis is preferably conducted by diagnosis ofprotein expression from the viewpoint of accurately diagnosing LSRpositive to realize a more optimal dosing. Protein expression may bediagnosed by using, for example, an anti-LSR antibody. In diagnosis ofan episode, an episode of LSR positive malignant tumor may be determinedto be present when a protein obtained from malignant tumor cells to betested derived from a patient is subjected to Western blot and a bandcorresponding to LSRs can be confirmed by visual inspection. Further, anepisode of LSR positive malignant tumor may be determined to be presentwhen the amount of LSR expression of malignant tumor cells derived froma patient is significantly larger relative to normal cells or LSRnegative malignant tumor cells. Further, an episode of LSR positivemalignant tumor may be determined to be present when total proteinobtained from malignant tumor cells derived from a patient and totalprotein obtained from normal cells or LSR negative malignant tumor cellsare subjected to Western blot and the malignant tumor cells derived fromthe patient have a significantly stronger band intensity correspondingto LSRs relative to the normal cells or LSR negative malignant tumorcells. Further, an episode of LSR positive malignant tumor may bedetermined to be present when serum or plasma obtained from malignanttumor patients and serum or plasma obtained form healthy individuals orLSR negative malignant tumor patients are subjected to ELISA usinganti-LSR antibodies and the amount of LSR expression is significantlymore for the serum or plasma derived from malignant tumor patientsrelative to healthy individuals or LSR negative malignant tumorpatients. The serum or plasma sample itself may be quantified, orexosomes may be isolated from the serum or plasma to subject LSRs in theexosomes to ELISA for analysis. RT-PCR may be used instead of Westernblot in such diagnosis for an episode of LSR positive malignant tumor.

The therapeutic method of malignant tumor according to one embodiment ofthe present invention may comprise a step of administering an LSRantagonist to a patient. Further, the method may comprise a step ofadministering an RNAi molecule directed to an LSR or a polynucleotideencoding the RNAi molecule to the patient.

One embodiment of the present invention is a novel diagnostic drug formalignant tumor, comprising an anti-LSR antibody. The diagnostic drugmay be, for example, a companion diagnostic drug for malignant tumortherapy targeting LSRs, comprising an anti-LSR antibody. Since there areLSR positive and LSR non-positive patients among malignant tumorpatients, therapeutic efficacy of the malignant tumor therapy targetingLSRs can be diagnosed if the companion diagnostic drug is used toinspect in advance whether malignant tumor is LSR positive. In suchdiagnosis, when the result is LSR positive, malignant tumor therapytargeting LSRs can be determined to be effective. “Companion diagnosis”in one embodiment of the present invention comprises diagnosisimplemented in order to assist in the optimal dosing by predictingindividual differences in the effect of agent or side effects forpatients by inspection. For clinical application of an antibodymedicament with anti-LSR antibodies, it is understood that selectivetherapy for LSR expressing-malignant tumor patients would lead toindividualized medicine. For this reason, a method of sorting out an LSRpositive patient is needed. An approach that inspects LSRs in a biopsytissue of cancer by an immunohistochemical staining method is consideredhighly useful as a method of sorting out an LSR positive patient.However, obtaining a biopsy tissue is highly invasive. Thus, a methodwith a low level of invasiveness is preferred. Furthermore, certaintypes of malignant tumor such as ovarian cancer are problematic in thatbiopsy tissue is difficult to obtain due to the issues related to thesite of cancer. In contrast, LSRs expressed in cancer or theextracellular domain thereof may be freely present in blood. In thisregard, the possibility of high level of LSR expression in ovariancancer tissue in a patient with a high blood LSR concentration issuggested when LSRs in the blood of a malignant tumor patient can bequantified. A blood sample is more advantageous than biopsy in that thelevel of invasiveness is low. It is highly likely that LSR is highlyexpressed in patient tissue with elevated blood LSR concentrationrelative to healthy individuals after quantifying the blood LSRconcentration by ELISA. Thus, measurement of blood LSR concentration isconsidered highly useful as a companion diagnostic drug.

A diagnostic drug for malignant tumor according to one embodiment of thepresent invention may be a diagnostic drug comprising an anti-LSRantibody for diagnosis of therapeutic efficacy of the anti-LSR antibodyor LSR antagonist on malignant tumor. Since there are LSR positive andLSR non-positive patients among malignant tumor patients, it is possibleto diagnose the therapeutic efficacy of an anti-LSR antibody or LSRantagonist to patients if the diagnostic agent is used in advance toinspect whether malignant tumor is LSR positive.

One embodiment of the present invention is a companion diagnostic methodfor malignant tumor therapy targeting an LSR, comprising inspectingwhether a malignant tumor sample of a malignant tumor patient is LSRpositive. Since there are LSR positive and LSR non-positive patientsamong malignant tumor patients, it is possible to diagnose thetherapeutic efficacy of malignant tumor therapy targeting an LSR if thecompanion diagnosis method is used to inspect in advance whethermalignant tumor is LSR positive. Such a diagnostic method may furthercomprise a step of isolating or extracting a malignant tumor sample of amalignant tumor patient. “Malignant tumor sample” in one embodiment ofthe present invention may be malignant tumor tissue or cells obtainedfrom a malignant tumor patient.

One embodiment of the present invention is a method of diagnosingprognosis of malignant tumor using an LSR expression level as anindicator. High level of LSR expression is demonstrated to be a poorprognosis marker (FIG. 38). Thus, a high level of LSR expression can beconsidered a poor prognosis marker. In one embodiment, cancer subjectedto prognosis is ovarian serous adenocarcinoma, but the cancer is notlimited thereto. It is understood that the present invention can beapplied to ovarian clear cell adenocarcinoma and the like. It is alsounderstood that the present invention can be applied to any other LSRpositive malignant tumor. A method of using such a (poor) prognosismarker may comprise, for example, a step of investigating whether LSRsare expressed in malignant tumor cells derived from a patient. Theprognosis (diagnosis) of LSR positive malignant tumor may be conducted,for example, by diagnosis or detection of mRNA expression or diagnosisor detection of protein expression. Such diagnosis or detection ispreferably conducted by diagnosis of protein expression from theviewpoint of accurately diagnosing LSR positive to realize a moreoptimal dosing. Protein expression may be diagnosed by using, forexample, an anti-LSR antibody. In diagnosis of an episode, the prognosisof malignant tumor may be determined to be poor when a protein obtainedfrom malignant tumor cells to be tested derived from a patient issubjected to Western blot and enhancement in a band corresponding toLSRs can be confirmed by visual inspection. Further, the prognosis ofmalignant tumor may be determined to be poor when the amount of LSRexpression of malignant tumor cells derived from a patient issignificantly larger relative to normal cells or LSR negative malignanttumor cells (e.g., LSR negative cell strains such as OVTOKO). Further,the prognosis of malignant tumor may be determined to be poor when totalprotein obtained from malignant tumor cells derived from a patient andtotal protein obtained from normal cells or LSR negative malignant tumorcells (e.g., LSR negative cell strains such as OVTOKO) are subjected toWestern blot and the malignant tumor cells derived from the patient havea significantly stronger band intensity corresponding to LSRs relativeto the normal cells or LSR negative malignant tumor cells (e.g., LSRnegative cell strains such as OVTOKO). Further, the prognosis ofmalignant tumor may be determined to be poor when serum or plasmaobtained from malignant tumor patients and serum or plasma obtained formhealthy individuals or LSR negative malignant tumor patients (e.g.,“patient whose LSR expression is negative in ovarian cancer tissue”) aresubjected to ELISA using anti-LSR antibodies and the amount of LSRexpression is significantly more for the serum or plasma derived frommalignant tumor patients relative to the healthy individuals or LSRnegative malignant tumor patients. The serum or plasma sample itself maybe quantified, or exosomes may be isolated from the serum or plasma tosubject LSRs in the exosomes to ELISA for analysis. RT-PCR may be usedinstead of Western blot in such diagnosis of an episode of LSR positivemalignant tumor.

One embodiment of the present invention is a method of inspectingtherapeutic efficacy of an anti-LSR antibody or LSR antagonist onmalignant tumor. The inspection method comprises, for example,inspecting whether a malignant tumor sample of a malignant tumor patientis LSR positive. The inspection method, which may comprise a step ofdetecting the presence of LSRs in a malignant tumor sample, may comprisea step of detecting that the amount of LSRs in the malignant tumorsample is significantly larger relative to normal cells or LSR negativemalignant tumor cells. For example, RT-PCR, Western blot, orimmunohistochemical staining method may be used in detecting LSRs. Thestandard of assessing the presence or absence of LSRs may be the same asthat in the aforementioned diagnosis of episode of LSR positivemalignant tumor. A method of inspecting therapeutic efficacy includes amethod of inspecting whether the method is effective for therapy.

One embodiment of the present invention is a suppressant for growth ofmalignant tumor cells, comprising anti-LSR antibodies. Further, it is amethod of suppressing growth of malignant tumor cells, comprisingcontacting anti-LSR antibodies with malignant tumor cells. Further, itis a suppressant for growth of malignant tumor cells, comprising an LSRantagonist. Further, it is a method of suppressing growth of malignanttumor cells, comprising contacting an LSR antagonist with malignanttumor cells. The therapeutic drug or suppressant for growth of malignanttumor cells according to the embodiment of the present invention may bean agent that reduces the growth rate, amount of growth, or volume ofmalignant tumor by 10, 20, 30, 40, 50, or 70% or more relative to a casewhere a therapeutic drug or growth suppressant is not added. Thepercentage may be within the range of two numerical values listed above.

One embodiment of the present invention is an agent for suppressing celldivision of malignant tumor cells, comprising an anti-LSR antibody.Further, it is a method of suppressing cell division of malignant tumorcells, comprising contacting an anti-LSR antibody with malignant tumorcells. Further it is an agent for suppressing cell division of amalignant tumor cell, comprising an LSR antagonist. Further, it is amethod of suppressing cell division of malignant tumor cells, comprisingcontacting an LSR antagonist with malignant tumor cells. The agent forsuppressing cell division of a malignant tumor cell according to theembodiment of the present invention may be an agent that reduces therate of malignant tumor cell division by 10, 20, 30, or 50% or morerelative to a case where an agent for suppressing cell division is notadded. The percentage may be within the range of two numerical valueslisted above.

One embodiment of the present invention is a therapeutic drug forLSR-dependent malignant tumor, comprising an anti-LSR antibody.LSR-dependent malignant tumor can be treated by using such a therapeuticdrug.

One embodiment of the present invention is use of an anti-LSR antibodyor LSR antagonist for producing a therapeutic drug for malignant tumor.In another embodiment, it is a use of an anti-LSR antibody formanufacturing a companion diagnostic drug for malignant tumor therapytargeting an LSR.

One embodiment of the present invention is a method of producing ananti-LSR antibody, comprising: introducing a polynucleotide encoding anLSR into a cell; expressing the LSR in the cell; and immunizing achicken with an antigen comprising a cell expressing the LSR. Accordingto the production method, an anti-LSR antibody that is excellent for thetreatment or diagnosis of LSR positive malignant tumor can beefficiently produced.

“Bond” in one embodiment of the present invention may be either acovalent bond or a non-covalent bond. For example, “bond” may be anionic bond, hydrogen bond, hydrophobic interaction, or hydrophilicinteraction.

(General Techniques)

Molecular biological approach, biochemical approach, and microbiologicalapproach used herein are well known and conventional approaches in theart that are described in, for example, Sambrook J. et al. (1989).Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and 3^(rd)Ed. thereof (2001); Ausubel, F. M. (1987). Current Protocols inMolecular Biology, Greene Pub. Associates and Wiley-Interscience;Ausubel, F. M. (1989). Short Protocols in Molecular Biology: ACompendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates and Wiley-Interscience; Innis, M. A. (1990). PCRProtocols: A Guide to Methods and Applications, Academic Press; Ausubel,F. M. (1992). Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, Greene Pub.Associates; Ausubel, F. M. (1995). Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub. Associates; Innis, M. A. et al. (1995). PCR Strategies,Academic Press; Ausubel, F. M. (1999). Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, Wiley, and annual updates; Sninsky, J. J. et al. (1999). PCRApplications: Protocols for Functional Genomics, Academic Press,Bessatsu Jikken Igaku [Experimental Medicine, Supplemental Volume],Idenshi Donyu Oyobi Hatsugen Kaiseki Jikken Ho [Experimental Methods forTransgenesis & Expression Analysis], Yodosha, 1997, and the like, therelevant portions (which can be the entire document) of which areincorporated herein by reference.

DNA synthesis techniques and nucleic acid chemistry for making anartificially synthesized gene are described in, for example, Gait, M. J.(1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press;Gait, M. J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRLPress; Eckstein, F. (1991). Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Adams, R. L. et al. (1992). The Biochemistry of theNucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). AdvancedOrganic Chemistry of Nucleic Acids, Weinheim; Blackburn, G. M. et al.(1996). Nucleic Acids in Chemistry and Biology, Oxford University Press;Hermanson, G. T. (1996). Bioconjugate Techniques, Academic Press and thelike, the relevant portions of which are incorporated herein byreference.

For example, as used herein, the oligonucleotide of the presentinvention can also be synthesized by a standard method known in the art,such as by using an automated DNA synthesizer (a synthesizercommercially available from Biosearch, Applied Biosystems or the like).For example, a phosphorothioate-oligonucleotide can also be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16: 3209), and amethyl phosphonate-oligonucleotide can also be prepared by using acontrol pore glass polymer support (Sarin et al., 1988, Proc. Natl.Acad. Sci. USA 85: 7448-7451).

As used herein, “or” is used when “at least one or more” of the matterslisted in the sentence can be employed. When explicitly described hereinas “within the range of two values”, the range also includes the twovalues themselves.

Reference literatures such as scientific literatures, patents, andpatent applications cited herein are incorporated herein by reference tothe same extent that the entirety of each document is specificallydescribed.

As described above, the present invention has been described whileshowing preferred embodiments to facilitate understanding. The presentinvention is described below based on Examples. The aforementioneddescription and the following Examples are not provided to limit thepresent invention, but for the sole purpose of exemplification. Thus,the scope of the present invention is not limited to the embodiments andExamples specifically described herein and is limited only by the scopeof claims.

EXAMPLES

Hereinbelow, the present invention is further described by examples.However, the present invention is not limited to these.

<Example 1> LSR Expression Analysis

1.1 Quantitative analysis of cell surface membrane protein according toiTRAQ™ method (Creative Proteomics, Shirley, N.Y.).

Identification of an ovarian-cancer-specific cancer antigen protein wastried by searching for a cell surface membrane protein highly expressedin ovary serous adenocarcinoma cell strains (OVCAR3, OVSAHO, and JHOS4)in comparison with normal ovarian epithelial cell strains (HOSE2C andE7/TERT). First, for a cell strain cultured in a 150 mm Petri dish, thecell surface membrane proteins were biotinylated withsulfo-NHS-SS-biotin. The extracted proteins were purified byNeurto-avidin beads. At this time, in order to correct the error betweenthe samples, sulfo-NHS-SS-biotin-labeled bovine serum albumin was addedto each in an equal amount as an internal standard, and was used forcorrection of quantification results by a mass spectrometer. Thepurified proteins were digested by trypsin and labeled with an iTRAQ™reagent. The samples were mixed into one and it was roughly fractionatedinto 24 fractions by ion exchange HPLC. Each of the fractions wasdesalinated and then measured by a mass spectrometer (nano LC-MS/MS)analysis. A data base was searched for the obtained data using proteomediscoverer ver. 1.1 and thereby the proteins were identified andquantified. It should be noted that the ovarian cancer surgery tissuesused in the examples were provided from patients whose agreements toinformed consents were obtained in Osaka University Hospital.

As a result of analysis according to an iTRAQ™ method, it was found thatLSR was specifically and highly expressed in the above-mentioned ovaryserous adenocarcinoma cell strains as below.

TABLE 1 E7TERT/ Description HOSE2C Criteria OVCAR3 OVSAHO JHOS4 LSR3.092 HOSE2C 10.939 11.412 9.073 E7/TERT 5.207 5.328 3.301

1.2 Rt-PCR

RNAs of a normal ovarian epithelial cell strain (HOSE2C), ovary serousadenocarcinoma cell strains (OVCAR3, OVSAHO, and JHOS4), ovarian clearcell adenocarcinoma cell strains (OVTOKO, OVMANA, OVISE, and RGMI),normal endometrial cell strains (E6/E7/TERT) and endometrial cancer(HEC1, HEC1A, HEC6, HEC88nu, HEC108, HEC116, HEC251, and SNGM) were eachpurified by the RNeasymini® kit (QIAGEN). Further, reverse transcriptioninto cDNA was carried out using the QuantiTect® Reverse TranscriptionKit (Qiagen). RT-PCR was carried out using the TaKaRaEx® Taq DNApolymerase (Takara Bio, Shiga, Japan). Primers of the followingsequences were used in the RT-PCR.

LSR:

forward primer (SEQ ID NO: 17) 5′-GGGAGGACCTCAGGGGTGGC-3′ andreverse primer (SEQ ID NO: 18) 5′-TGGTGGGGGTGGGGTCTTGG-3′; and β-actin:forward primer (SEQ ID NO: 19) 5′-AGCCTCGCCTTTGCCGA-3′ andreverse primer (SEQ ID NO: 20) 5′-CTGGTGCCTGGGGCG-3′.

The results of the above were shown in FIGS. 1 to 3. In the ovary serousadenocarcinoma cell strains OVCAR3, OVSAHO, and JHOS4, the ovarian clearcell adenocarcinoma cell strains OVMANA, OVISE, and RGMI, and theendometrial cancer cell strains HEC1, HEC1A, HEC6, HEC88nu, HEC108,HEC116, HEC251, and SNGM, a band corresponding to LSR mRNA was detected.In the normal ovarian epithelial cell strains, for HOSE2C, it was notdetected.

1.3 Western Blot

Ten μg each of proteins obtained from the normal ovarian epithelial cellstrain (HOSE2C), the ovary serous adenocarcinoma cell strains (OVCAR3,OVSAHO, SKOV3, JHOS2, and JHOS4), the ovarian clear cell adenocarcinomacell strains (OVTOKO, OVMANA, OVISE, and RGMI), the normal endometrialcell strains (E6/E7/TERT) and the endometrial cancer (HEC1, HEC1A, HEC6,HEC88nu, HEC108, HEC116, HEC251, and SNGM) were applied to SDS-PAGE (5to 20% gradient gel (Wako Pure Chemical Industries, Ltd.)). Then, theywere subjected to migration at 40 mA for 50 minutes and subsequentlytransferred to PVDF membranes at 120 mA for 1 hour. After the transfer,blocking was carried out in 1% BSA/TBST (TBS+0.1% Tween 20) at roomtemperature for 1 hour and then incubation with an anti-LSR antibody(Santa Cruz Biotechnology) was carried out at room temperature for 1hour. After washing with TBST for 10 minutes three times for each, thePVDF membranes were incubated at room temperature for 1 hour using anHRP-labeled anti-rabbit antibody (GE healthcare) that had been diluted5,000 times with TBST. The PVDF membranes were washed with TBST for 10minutes three times for each and then the reacted proteins were detectedby a fluorescence reaction system (Perkin Elmer, Inc.).

The results of the above are shown in FIGS. 4 to 6. In the ovary serousadenocarcinoma cell strains OVCAR3, OVSAHO, and JHOS4, ovarian clearcell adenocarcinoma cell strains OVMANA, OVISE, and RGMI, and theendometrial cancer cell strains HEC1, HEC1A, HEC6, HEC88nu, HEC108,HEC116, HEC251, and SNGM, a band corresponding to LSR was detected.Meanwhile, in the ovarian clear cell adenocarcinoma cell strain OVTOKO,a band corresponding to LSR was not detected. In the normal ovarianepithelial cell strain, a band corresponding to LSR was not detected inany of HOSE2C. From the above results, it is understood that the LSRprotein is specifically expressed at the above-mentioned cancers.

Moreover, for proteins obtained from normal ovarian tissues, ovaryserous adenocarcinoma surgery tissues, ovarian clear cell adenocarcinomasurgery tissues, normal endometrial tissues, and endometrial cancersurgery tissues, Western blot was carried out using an anti-LSR antibody(Santa Cruz Biotechnology). An anti-GAPDH antibody (Santa CruzBiotechnology) was used as a loading control. The tissues used for theWestern blot were obtained from healthy humans or patients sufferingfrom respective cancers.

The results of the above are shown in FIGS. 7 and 8. The black circlesin the figures mean that the expression of LSR was confirmed. In thenormal ovarian tissue and the normal endometrial tissue, LSR was notexpressed. In the ovary serous adenocarcinoma tissue, LSR wasspecifically expressed in 13/16 people (81%). In the ovarian clear celladenocarcinoma surgery tissue, LSR was specifically expressed in 4/11people (36%). In the endometrial cancer tissue, LSR was specificallyexpressed in 19/35 people (54%). From these results, it was found thatwhile LSR positive patients were present in ovarian cancer patients anduterine cancer patients, a certain number of LSR negative patients werealso present.

<Example 2> Making and Evaluation

2.1 Making of Human LSR-Expressing Chicken Cell Strain and Immunizationto Chicken

cDNA (SEQ ID NO: 7) of human LSR was cloned to a mammalian expressionvector (pcDNA3.1-V5/His-TOPO) to make a LSR expression vector. This LSRexpression vector encodes a fused protein in which a V5/His tag wasfused to the C-terminal of the human LSR. Then, the LSR expressionvector was transfected into a chicken lymphoblast-like cell strainaccording to an electroporation method and then 2 mg/ml of G418 wasadded to select an expression cell. Chicken was hyperimmunized with theobtained LSR-expressing cell strain. An antibody titer was measured byflow cytometry (FACS) analysis. With regard to the FACS analysis, theprotocol of FACSCalibur (BD, USA) was followed.

2.2 Making of scFv Phage Antibody Library from Immunized Chicken Spleen

The spleen was extracted from the immunized chicken and then thelymphocytes were separated. The RNA was extracted from the obtainedlymphocytes, a cDNA was synthesized, and a scFv phage antibody librarywas made. For the making of a scFv phage antibody library, a techniquedescribed in “Nakamura et al., J Vet Med Sci. 2004 July; 66(7): 807-14”was followed.

2.3 Panning Selection

The scFv phage antibody library was added to a non-LSR-expressing cellstrain to carry out absorption operation of nonspecific phages, and thenwas reacted with a LSR-expressing cell strain. In Lot 1, a mammaliancell strain was used and in Lot 2, cell panning was carried out usingthe chicken lymphoblast-like cell strain used for immunization. Afterwashing with organic solvent, phages specifically binding to theLSR-expressing cell strain were recovered and then Escherichia coli wereinfected with it. Panning was carried out four times and then thereactivity of the library was confirmed by FACS analysis using theLSR-expressing cell strain. Phages from a library of which thereactivity had most increased were cloned, positive clones wereselected, and then the sequences of six types of clones were determined(SEQ ID NOs: 1 to 6 and FIG. 9). For cell panning, a method described in“Giordano et al., Nat Med. 2001 November; 7(11): 1249-53” was followed.

2.4 Recombination to Recombinant Mouse/Chicken Chimeric (IgG2a) Antibody

The VH and VL in a chicken-derived antibody gene were PCR amplifiedusing a DNA strand encoding a scFv phage antibody as a template and thenwere cloned into a mouse/chicken chimeric (IgG2a) expression vector (Hchain: pcDNA3.1 and L chain: pcDNA4 (Invitrogen)). The made construct ofthe H chain and the L chain was transfected into mammalian culture cellsand then expressed antibodies (anti-LSR mouse/chicken chimericmonoclonal antibodies) were purified using Protein G Sepharose (GE).From the above, six types of clones of anti-LSR antibodies (#9-7, #16-6,No. 26-2, No. 27-6, No. 1-25, and No. 1-43) were obtained. Forrecombination, a technique described in “Tateishi et al., J Vet Med Sci.2008 April; 70(4): 397-400” was followed.

2.5 Evaluation of Reactivity to Various Ovary Cancer Cell Strains

Using five types (#1-25, #9-7, #16-6, No. 26-2, and No. 27-6) from theanti-LSR antibodies obtained in 2.4 described above, the reactivity tovarious ovary cancer cell strains was investigated by FACS analysis. Theresults are shown in FIGS. 10 to 14. In ovary serous adenocarcinoma cellstrains (OVSAHO and JHOS2) and ovarian clear cell adenocarcinoma cellstrains (RGM-I and OVISE), a significant shift difference was observedby the presence or absence of the anti-LSR antibodies.

Immunohistochemical Staining

For tissues of ovarian cancer (84) cases, the expression of LSR wasanalyzed by immunohistochemical staining. A primary antibody from CloudClone Corp. (PAD744Hu01) was used and the Dako ChemMate™ ENVISION™Kit/HRP (DAB)-universal kit (K5007) was used to carry out staining.

Results of the immunohistochemical staining were rated with scores. Thescore rating was on a five-point scale: 0+(no staining cell); 1+(palestaining in any proportion of cells); 2+(darkly staining cells (<25% ofarea)); 3+(darkly staining cells (25 to 49% of area); and 4+(darkstaining (>50% area)). Scores 0, 1, and 2 were classified as a LSR lowexpression group and scores 3 and 4 were classified as a LSR highexpression group. They were classified into the groups of the LSR lowexpression group and the LSR high expression group, a survival curve wascreated using the Kaplan-Meier method, and a log-rank test was carriedout.

Consequently, in ovary serous adenocarcinoma, it became clear thatprognosis in the LSR high-expression cases was significantly worse thanthat in the low-expression cases (median OS: 73.8 vs 105.5 months)(p=0.0293). Meanwhile, although a significant difference is notrecognized in ovarian clear cell adenocarcinoma, it was observed thatprognosis in the LSR high-expression cases tended to be worse than thatin the low-expression cases (median OS: 71.4 vs 87.4 months) (p=0.1362).In each of ovary serous adenocarcinoma and ovarian clear celladenocarcinoma, the expression of LSR was examined for surgery tissuesof lymph node metastasis sites and greater omentum metastasis sitesaccording to an immunohistochemical staining method and consequently itwas confirmed that LSR is expressed at cancer tissues of the metastasissites.

As shown in FIG. 29, previously, there was no effective therapeuticmethod for recurrent ovarian cancer. Conventionally, there was noeffective therapeutic method for recurrent ovarian cancer. Theepidemiological characteristic of ovarian cancer is that ovarian cancerreadily infiltrate into the surrounding by the lymph node and peritonealmetastasis or the like and advances quickly. For instance, 40% or moreof ovarian cancer in Japanese patients is considered serous, 24% clearcells, 17% endometrioid, and 13% mucinous adenocarcinoma. As a 1st lineof defense, cisplatin or taxol is used, and Avastin is used forrecurrent ovarian cancer. However, it was considered that improvement insurvival rate was not observed. Antibody pharmaceutical productsapproved as a therapeutic drug for cancer include those shown in thetable of FIG. 29 (Carter P J Nat. Rev. Immunol. 006, May 6(5)343-357,Review). Since there is no therapeutic method for the advanced stage andthe time of recurrence, ovarian cancer is presumed to be poor prognostictumor and the development of a novel therapeutic method is presumedurgent. For example, as shown in FIG. 29, the five year survival rate inStage IV was 31% (Japan Society of Obstetrics and Gynecology, Fujinkashuyou iinkai houkoku [Gynecology tumor committee report], 2012, vol.64, No. 6).

In this regard, the inventors confirmed as shown in FIG. 30 whether LSRis expressed at the ovarian cancer primary site. The protocol of it isas below. It should be noted that a technique similar to theabove-mentioned technique in the present example was used inimmunostaining of LSR. The expression of LSR was analyzed byimmunohistochemical staining. A primary antibody from Cloud Clone Corp.(PAD744Hu01) was used and the Dako ChemMate™ ENVISION™ Kit/HRP(DAB)-universal kit (K5007) was used to carry out staining.

In addition, using proteins extracted from ovarian cancer surgerytissue, the expression of LSR was examined according to the Western blotmethod. Consequently, it became clear that LSR is more highly expressedin ovarian clear cell adenocarcinoma and ovary serous adenocarcinomathan normal ovarian tissue. GAPDH indicated a control group.

The result is shown in FIG. 30. As shown in FIG. 30, it was confirmedthat LSR was expressed at the ovarian cancer primary site.

In addition, it was confirmed whether it was expressed at metastasissites other than the primary site. The protocol of it is shown as below.It should be noted that a technique similar to the above-mentionedtechnique in the present example was used in immunostaining of LSR.Specifically, the expression of LSR was analyzed by immunohistochemicalstaining. A primary antibody from Cloud Clone Corp. (PAD744Hu01) wasused and the Dako ChemMate™ ENVISION™ Kit/HRP (DAB)-universal kit(K5007) was used to carry out staining.

The results are shown in FIGS. 31 to 32. As shown in these figures, theyindicate that it is also expressed at metastasis sites other than theprimary site. From these facts, it is understood for the presentinvention that a LSR antibody medicine can be expected to exhibit anantitumor effect on not only primary ovarian cancer but also metastasissites.

Then, for the expression of LSR, it was confirmed whether LSR is alsoexpressed in other cells (FIGS. 33 to 35). These include expression atearly ovarian clear cell adenocarcinoma, and gastric cancer andsignet-ring cell cancer of gastric cancer, which are adenocarcinomaother than ovary cancer. Immunohistochemical staining of LSR was carriedout according to a similar technique to the above-mentioned.Specifically, the expression of LSR was analyzed by immunohistochemicalstaining. A primary antibody from Cloud Clone Corp. (PAD744Hu01) wasused and the Dako ChemMate™ ENVISION™ Kit/HRP (DAB)-universal kit(K5007) was used to carry out staining.

The results are shown in FIGS. 33 to 35. As shown in FIG. 33, itindicates that LSR is also expressed at early ovarian clear celladenocarcinoma. As shown in FIG. 34, it indicates that LSR is alsoexpressed at gastric cancer as adenocarcinoma other than ovarian cancer.In addition, as shown in FIG. 35, it indicates that LSR is alsoexpressed at signet-ring cell cancer of gastric cancer. From these, itis understood that it can be used in the therapy for ovarian cancer evenin the early stage and it has therapeutic possibility for otheradenocarcinoma such as gastric cancer and the like.

Then, poor prognosis was investigated. In order to confirm whetherhighly-LSR-expressing ovary serous adenocarcinoma has a poor prognosisin comparison with a low expression group, the prognosis of ovary serousadenocarcinoma patients and ovarian clear cell adenocarcinoma patientswas investigated based on being high or low in the expression of LSR.For ovary serous adenocarcinoma, 21 cases of strongly-LSR-expressingpatients and 12 cases of weakly-expressing patients were investigatedand for ovarian clear cell adenocarcinoma, 27 cases ofstrongly-LSR-expressing patients and 24 cases of weakly-expressingpatients were investigated. The result is shown in FIG. 38. As shown inFIG. 38, it was found that the highly-LSR-expressing ovary serousadenocarcinoma has poor prognosis in comparison with the low expressiongroup.

(Epitope Analysis)

PepStar™ peptide microarrays were made on glass slides obtained from JPTPeptide Technologies (GmbH). Fifteen-mer overlapping peptides thatoverlap to the extracellular domain region of LSR by 10 amino acids weresynthesized and solid-phased to glass slides. Binding of a purifiedrecombinant antibody to a peptide was carried out according to theinstructions, however, it included changes to some parts (www.jpt.com).A primary antibody was reacted at a concentration of 1.0 μg/mL and theglass slides were washed with TBST (50 mM TBS-buffer including 0.1%Tween20, pH 7.2). Then, it was reacted using Cy5-labeled goatanti-chicken IgY (Jackson Immuno Research) and washed with TBST fivetimes and the glass slides were washed with ddH2O five times. The glassslides were dried by spraying argon gas mildly. A fluorescence signalwas detected using the GenePix® 4200AL scanner (Molecular Devices) atresolution of 10 μm.

The result is shown in FIG. 39. As shown in FIG. 39, it became clearthat there are two types of epitopes and amino acids 116 to 135 (towhich the antibodies #9-7, 1-25, 16-6, 26-2, and 1-43 correspond) andamino acids 216 to 230 (to which the antibody #27-6 corresponds) arepresent.

(Cross Reaction)

In order to investigate that an anti-LSR antibody cross-reacts withmouse LSR, a mouse LSR expression vector or a control vector wastransfected into COST cells and the reactivity with various clones ofanti-LSR antibodies prepared in the present example was analyzed byFACS®. The result is shown in FIG. 40. Consequently, it became clearthat all the clones exhibit cross reaction with mouse LSR. The fact thatan anti-LSR antibody cross-reacts with mouse LSR as described abovemakes it possible to use mice as an animal for a safety test. Pleaserefer to FIG. 52 and thereafter, where actual acute toxicity tests werecarried out.

2.6 Expression Analysis of LSR by Immunohistochemical Staining Method

Slices of paraffin-embedded tissues of ovary serous adenocarcinomatissue and endometrial cancer tissue were deparaffinization-treated anddehydrated with alcohol. Then, using an anti-LSR antibody (#1-25 or#9-7), immunohistochemical staining for LSR was carried out according tothe ABC method. The results are shown in FIGS. 15 to 17. In the ovaryserous adenocarcinoma tissue and the endometrial cancer tissue, LSR washighly expressed at the tumor sites.

Immunohistochemical Staining Using Normal Frozen Tissue Array

FDA standard human tissue microarrays (T6234701-2, Biochain) wereimmunohistochemically stained using the anti-LSR antibody #1-25. Theexpression of LSR was observed at liver and testis of various normaltissues (FIGS. 36 and 37).

Calculation of Binding Constant

Various concentration of various anti-LSR antibodies were reacted withRMG-I cells and they were stained using goat anti-mouse IgG-FITC(Southern Biotech, Birmingham, Ala., USA) and analyzed by a FACSCanto®II cytometer (Becton Dickinson). With regard to the fluorescenceintensity of FITC, a KD value was analyzed using GraphPad Prism®Software Version 6.0 for Windows (GraphPad Software Inc., San Diego,Calif., USA). The result is shown in FIG. 37B. When the binding abilityof the made LSR antibodies was analyzed by FACS®, antibodies having ahigh binding ability were obtained. The following analysis was carriedout using two kinds of clones having the highest binding ability in them(FIG. 37B). For #9-7, K_(D)=2.52 nM; for #1-25, K_(D)=2.03 nM; for#16-6, K_(D)=2.33 nM; for #26-2, K_(D)=4.04 nM; for #27-6, K_(D)=4.29nM; and for #1-43, K_(D)=24.62 nM.

Cell Cycle Analysis

Ovarian clear cell adenocarcinoma (RMG-I) was seeded into a 6-well plateat 15,000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The culture supernatant was discarded and 100 μg/ml anti-LSRantibody or mouse IgG2a diluted with RPMI 1640 medium (containing 1% FBSand 1% penicillin-streptomycin) was added at 2 mL/well for each. After96 hours, cell cycle analysis was carried out using the Cycle Test Plus®DNA Reagent kits (BD Biosciences).

Western Blot

Ovarian clear cell adenocarcinoma (RMG-I) was seeded into a 6-well plateat 15,000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The culture supernatant was discarded and 100 μg/ml anti-LSRantibody or mouse IgG2a diluted with RPMI 1640 medium (containing 1% FBSand 1% penicillin-streptomycin) was added at 2 mL/well for each. After96 hours, proteins were extracted using a RIPA buffer (10 mM Tris-HCl,pH 7.5, 150 mM NaCl, 1% Nonidet® P-40, 0.1% sodium deoxycholate, 0.1%SDS, 1× phosphatase inhibitor cocktail (Nacalai Tesque), and 1× proteaseinhibitor cocktail (Nacalai Tesque)) and the difference in proteinexpression was analyzed by the Western blot method. The followingantibodies were used as primary antibodies: anti-LSR antibody(sc-133765) and anti-GAPDH antibody (sc-25778) (Santa Cruz Biotechnology(Santa Cruz, Calif.)); and anti-cyclin D1 antibody (#2926), anti-p27antibody (#3686), anti-phospho-Rb (Ser780) antibody (#9307),anti-phospho-Rb antibody (Ser807/811) (#9308), anti-Rb antibody (#9313),anti-phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) antibody (#4370),anti-p44/42 MAPK (Erk1/2) antibody (#4695), anti-phospho-MEK1/2(Ser217/221) antibody (#9154), and anti-MEK1/2 antibody (#9126) (CellSignaling Technology).

<Example 3> Growth Suppression of LSR Positive Malignant Tumor 3.1Growth Inhibition Assay with Anti-LSR Antibody

Ovarian clear cell adenocarcinoma (RMG-I) was seeded into a 96-wellplate at 1000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The cell supernatant on the 96-well plate was discarded anddiluted solutions (0 μg/ml, 1 μg/ml, 10 μg/ml, and 100 μg/ml) of ananti-LSR antibody (#9-7 or #1-25) were each added at 100 μL/well. After72 hours, cell growth assay was carried out according to the TWT-8 assaymethod. In addition, mouse IgG2 (Biolegend, Inc., 400224, MOPC-173),which is non-anti-LSR antibody, was used as a control. The results areshown in FIGS. 18 and 19. By contacting an anti-LSR antibody, the growthof ovarian cancer cells (RMG-I) was suppressed.

Ovarian clear cell adenomatous cancer (A2780) was seeded into a 96-wellplate at 1000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The cell supernatant on the 96-well plate was discarded anddiluted solutions (1 μg/ml, 10 μg/ml, and 100 μg/ml) of an anti-LSRantibody (#9-7 or #26-2) were each added at 100 μL/well. After 72 hours,cell growth assay was carried out according to the TWT-8 assay method.In addition, mouse IgG2 (Biolegend, Inc., 400224, MOPC-173), which isnon-anti-LSR antibody, was used as a control. The result is shown inFIG. 20. By contacting an anti-LSR antibody, the growth of ovariancancer cells (A2780) was suppressed.

Cell Cycle Analysis

Ovarian clear cell adenomatous cancer (RMG-I) was seeded into a 6-wellplate at 15,000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The cell supernatant on the 6-well plate was removed and ananti-LSR antibody (#1-25) that had been diluted to a concentration of100 μg/ml with RPMI 1640 medium (containing 1% FBS and 1%penicillin-streptomycin) was added at 2 mL/well for each. In addition,mouse IgG2 (Biolegend, Inc; 400224, MOPC-173), which is a non-anti-LSRantibody, was used as a control. Ninety six hours after the addition ofthe antibody, intracellular DNA was stained using the Cycle Test Plus®DNA Reagent kits (BD Biosciences) and cell cycle analysis was carriedout using a FACSCanto® flow cytometer.

The result is shown in FIG. 41. It was recognized that by contacting theanti-LSR antibody, the S phase and the G2/M phase in the cell cycle ofovarian cancer cells (RMG-I) were significantly decreased and the G0/G1phase was significantly increased in comparison with the controlantibody-treatment group.

Western Blot Analysis

Ovarian clear cell adenomatous cancer (RMG-I) was seeded into a 6-wellplate at 15,000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The cell supernatant on the 6-well plate was removed and ananti-LSR antibody (#1-25) that had been diluted to a concentration of100 μg/ml with RPMI 1640 medium (containing 1% FBS and 1%penicillin-streptomycin) was added at 2 mL/well for each. In addition,mouse IgG2 (Biolegend, Inc., 400224, MOPC-173), which is a non-anti-LSRantibody, was used as a control. Seventy two hours after the addition ofthe antibody, proteins were extracted, the expression variation ofproteins related to the cell cycle was analyzed by the Western blotmethod using the following various antibodies: anti-cyclin D1 (#2926),anti-p27 (#3686), anti-phospho-Rb (Ser780) (#9307), anti-phospho-Rb(Ser807/811) (#9308), anti-Rb (#9313), anti-phospho-p44/42 MAPK (Erk1/2)(Thr202/Tyr204) (#4370), anti-p44/42 MAPK (Erk1/2) (#4695),anti-phospho-MEK1/2 (Ser217/221) (#9154), and anti-MEK1/2 (#9126) (CellSignaling Technology).

The result is shown in FIG. 42. It was recognized that by contacting theanti-LSR antibody, the expression of Cyclin D1 was decreased and theexpression of p27 was increased in the ovarian cancer cells (ovariancancer cells (RMG-I)) in comparison with the control antibody. Moreover,it was recognized that the phosphorylation levels of phospho-Rb (Ser780)(#9307) and phospho-Rb (Ser807/811) were decreased. It was alsorecognized that the phosphorylation levels of phospho-MEK1/2(Ser217/221) and phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) as kinasesrelated to cell growth were decreased.

The above result indicates that the anti-LSR antibody directlysuppressed the growth of malignant tumor cells, and it was a surprisingresult. As the mechanism of this, for example, it is thought that theanti-LSR antibody binds to a malignant tumor cell, thereby a cell clumpof malignant tumor cells was formed, and consequently the cell divisionwas suppressed.

3.2 Epitope Analysis

For the anti-LSR antibodies obtained in 2.4 described above, epitopeswere analyzed as mentioned above and specifically identified. Inaddition, a growth suppressing effect on LSR positive malignant tumor isinvestigated for the anti-LSR antibodies for which the epitopes wereidentified. Consequently, it is understood that an anti-LSR antibodyrecognizing a specific epitope significantly suppresses the growth ofLSR positive malignant tumor in comparison with anti-LSR antibodiesrecognizing other epitopes.

3.3 Growth Inhibition Assay with siRNA

Ovary serous adenocarcinoma cells (OVSAHO) were seeded into a 96-wellplate at 1000 cells/well and incubated in a CO₂ incubator at 37° C.overnight. The cell supernatant on the 96-well plate was discarded andthe siRNA was transfected with LIPOFECTAMINE® 2000. After 120 hours,cell growth assay was carried out according to the WST-8 assay method.LSR siRNA and negative control siRNA were obtained from QIAGEN. The LSRsiRNA has a RNA sequence complementary to LSR mRNA (LSR siRNA 1: SEQ IDNO: 9 and LSR siRNA 2: SEQ ID NO: 10). The result is shown in FIG. 21.By contacting the LSR siRNA, the growth of ovarian cancer cells (OVSAHO)was suppressed.

3.4 Relation to Lipid Metabolism

It is confirmed whether the uptake of lipids (cholesterol) is elevatedin stably LSR-expressing cells described in the Examples. Afterconfirming whether the VLDL metabolism is elevated, it was confirmedwhether the elevation in metabolism due to VLDL is inhibited byadministration of a LSR antibody.

The protocol is shown as below.

Lipids were quantified using an empty vector strain of SKOV3 (EMP1) anda LSR-forcibly expressing strain (L45). In a low concentration, theywere seeded at 5×10⁵ cells per 10 cm dish and, in a high concentration,at 5×10⁵ cells per 10 cm dish and were cultured for 48 hours. Themediums were not exchanged. By adding a methanol+chloroform mixturesolution to cells suspended in PBS and centrifuging it, an organic layeras the lower layer was recovered to extract lipids. Lipids werequantified using LabAssay™ Triglyceride (GPO/DAOS method, Wako PureChemical Industries, Ltd.), LabAssay™ Cholesterol (cholesteroloxidase/DAOS method, Wako Pure Chemical Industries, Ltd.), andPhospholipid C-test WAKO™ (choline oxidase/DAOS method, Wako PureChemical Industries, Ltd.). The metabolism elevation due to VLDL wasmeasured using the Extracellular Flux Analyzer XFe24™ (PrimetechCorporation). Assay was carried out after glucose in the buffer wasremoved, glutamine was added, and the antibody amount was increased from10 ug/ml to 100 ug/ml.

Consequently, on day 1, the high was conf: 100% and the Low was 50 to60%. On day 2, the High was 100% and the Low was 70%.

The results are shown in FIGS. 22 to 24. As shown in FIG. 22, the uptakeof lipids (cholesterol) was elevated in the stably LSR-expressing cellsdescribed in the Examples. As shown in FIG. 23, the uptake of lipids(cholesterol) in high-density culture was elevated in the stablyLSR-expressing cells described in the Examples. As shown in FIG. 24,although the LSR expression described in the Examples make the VLDLmetabolism elevated, the elevation of metabolism due to VLDL wasinhibited by administration of a LSR antibody (#9-7). In #1-25, althoughthe inhibition of metabolism elevation was observed in some degree, thedegree was less than #9-7. While not wishing to be bound by theory, thisdifference is believed due to the difference in epitope recognitionsites depending on the clones.

<Example 4> Analysis of Antitumor Effect in Mouse by Anti-LSR MonoclonalAntibody

An ovarian clear cell adenomatous cancer cell strain RMG-I wassubcutaneously implanted to Scid mice (6-week old, female) at 1×10⁶cells/100 μl (PBS:Matrigel®=1:1). On day 14 after the implantation, themice were divided into two groups and an anti-LSR antibody (#1-25) or anisotype control antibody (Mouse IgG2a, M7769, Sigma) wasintraperitoneally administered at 10 mg/kg at a frequency of twice aweek and a total of 6 times (FIG. 25). The RMG-I-implanted mice weredissected on day 25 after the start of the antibody administration, andthe tumor weight was also measured. The following was calculated: tumorvolume=major axis×minor axis×height.

As a result of measuring a tumor volume, a significantly inhibitoryeffect on tumor growth in vivo was exhibited in the anti-LSR antibodyadministered group relative to the control IgG administered group (FIGS.26 to 28). A significant difference in tumor weight was also recognized.

<Example 5> Analysis of Antitumor Effect in Mouse by Anti-LSR MonoclonalAntibody

An ovarian clear cell adenomatous cancer cell strain RMG-I wassubcutaneously implanted to NOD/Scid mice (6-week old, female) at 1×10⁶cells/100 μl (PBS:Matrigel®=1:1). On day 14 after the implantation, themice were divided into two groups and an anti-LSR antibody (#1-25) or anisotype control antibody (Mouse IgG2a, M7769, Sigma) wasintraperitoneally administered at 10 mg/kg at a frequency of twice aweek and a total of 6 times (FIG. 43). The RMG-I-implanted mice weredissected on day 25 after the start of the antibody administration, andthe tumor weight was also measured. The following was calculated: tumorvolume=major axis×minor axis×height.

As a result of measuring a tumor volume, in the NOD/Scid mice, asignificantly inhibitory effect on tumor growth in vivo was exhibited inthe anti-LSR antibody administered group relative to the control IgGadministered group (FIG. 44). A significant difference in tumor weightwas also recognized. In addition, as a result of immunohistochemicallystaining a tumor tissue with an anti-Ki-67 antibody, a significantdecrease in the number of Ki-67 positive cell was recognized in theanti-LSR antibody administered group in comparison with the control IgGadministered group. From this, it became clear that the anti-LSRantibody exhibits activity of inducing the arrest of the cell cycle invivo (FIG. 45).

<Example 6> Analysis of Antitumor Effect in Mouse by Anti-LSR MonoclonalAntibody

SKOV3-L45 in which a LSR negative ovary serous adenocarcinoma cellstrain SKOV3 was made stably express LSR, or SKOV3-E1 into which anempty vector had been gene-transferred was subcutaneously implanted toScid mice (6-week old, female) at 5×10⁵ cells/100 μl(PBS:Matrigel®=1:1). On day 14 after the implantation, the mice weredivided into two groups and an anti-LSR antibody (#1-25) or an isotypecontrol antibody (Mouse IgG2a, M7769, Sigma) was intraperitoneallyadministered at 10 mg/kg at a frequency of every other day and a totalof 8 times (FIG. 46). The mice were dissected on day 18 after the startof the antibody administration, and the tumor weight was also measured.The following was calculated: tumor volume=major axis×minor axis×height.

As a result of measuring a tumor volume, in the SKOV3-L45-implanted Scidmice, a significantly inhibitory effect on tumor growth in vivo wasexhibited in the anti-LSR antibody administered group relative to thecontrol IgG administered group (FIG. 47). Meanwhile, in the tumor volumeof the mice into which the LSR negative SKOV3-E1 had been implanted, asignificant difference was not recognized (FIG. 47). A similar resultwas also obtained in tumor weight (FIG. 48). From this, it was suggestedthat in order for an anti-LSR antibody to exhibit an antitumor effect,it is necessary that LSR is expressed at tumor cells.

Tumors of the SKOV3-L45-implanted Scid mice and the SKOV3-E1-implantedScid mice were extracted and fat droplets were observed byelectronmicroscopy. Consequently, in the SKOV3-L45-implanted tissue,many accumulations of fat droplets were recognized in comparison withthe SKOV3-E1-implanted tumor tissue (FIG. 49). In the stablyLSR-expressing cell strain and the control-vector-expressing cellstrain, when fat droplets after the VLDL administration were compared,it was recognized that fat droplets in the LSR-expressing cell strainare larger and the number thereof is greater.

In order to investigate that an anti-LSR antibody binds to LSR andexhibits activity of internalizing in a cell, an antibody labeling wasprepared using CypHer® 5E mono NHS ester dye (GE Healthcare) and wasused in an assay. A LSR positive cell strain SKOV3-L45 cell strain and aLSR negative cell strain SKOV3-E1 and a CypHer® 5E-labeled antibody wereincubated for 3 hours and they were observed by the In cell analyzer2000. Consequently, all the clones of #9-7, #1-25, #16-6, #26-2, and#27-6 exhibited activity of internalizing (FIGS. 50 and 52). FIG. 50shows that if an anticancer agent is conjugated to an antibody, it isapplicable as an antibody-drug conjugate (ADC). FIG. 51 shows that if ananticancer agent is conjugated to an antibody, it is applicable as anantibody-drug conjugate (ADC).

These experiments were carried out as below. Tumor tissues wereextracted from subcutaneously-RMG-I-implanted NOD/SCID mice to which acontrol antibody or the anti-LSR antibody #1-25 has been administered.From the tumor tissues, formalin fixed paraffin embedded blocks weremade and the expression of Ki67 was analyzed by immunohistochemicalstaining. A primary antibody from Leica Biosystems Inc. (NCL-L-Ki67-MM1)was used and staining was carried out using the Dako ChemMate™ ENVISION™Kit™/HRP (DAB)-universal kit (K5007). As a result of calculating theproportion of Ki-67 positive cells in the respective visual fields, asignificant decrease in the proportion of Ki-67 positive cells wasrecognized in the anti-LSR antibody #1-25 administered group incomparison with the control antibody administered group. From this, itwas suggested that by administering the anti-LSR antibody #1-25, thecell cycle arrest is also induced in vivo.

Example 7: Safety Test

Then, a safety test was carried out for an antibody of the presentinvention. Since the anti-LSR antibody #1-25 also exhibits crossreaction with mouse LSR, an acute toxicity test in the case ofadministration to a mouse was carried out. One mg of Mouse IgG2a (Sigma,M7769) or the anti-LSR antibody #1-25 was intraperitoneally administeredto each of male and female C57BL/6J (8w) mice, the mice were dissectedon day 7, the brain, heart, kidney, liver, lung, and spleen wereextracted, and pathological analysis by HE staining was carried out. Inaddition, the blood was collected and analyzed using an automated bloodcell counting device (VetScan® HMII) and a biochemical blood analyzerfor animal (VetScan® VS2) (FIG. 52). Consequently, in the data of bloodcell number, any significant change was not recognized in the both(FIGS. 53 and 54). Similarly, in the blood biochemical data, anysignificant change was not recognized in the both (FIGS. 55 and 56).From this, it is understood that the anti-LSR antibody #1-25 has lowtoxicity and high safety.

The above Examples 1 to 4 show the following: (i) on contacting ananti-LSR antibody with malignant tumor cells, the growth of themalignant tumor cells is suppressed; (ii) on making a LSR antagonist acton malignant tumor cells, the growth of the malignant tumor cells issuppressed; (iii) by administering an anti-LSR antibody to a malignanttumor patient, the therapy for malignant tumor can be carried out; (vi)while LSR positive patients were present in malignant tumor patients, acertain number of LSR negative patients were also present; (v) inmalignant tumor therapy in which LSR is targeted, it is important todiagnose whether LSR positive is present or absent in a malignant tumorpatient before the therapy; and the like.

As above, the present invention is described based on the examples.These examples are only illustrations and those skilled in the art willunderstand that various variations are possible and such variations alsofall within the scope of the present invention.

As described above, the present invention is illustrated by preferableembodiments of the present invention. However, it will be understoodthat the scope of the present invention should be interpreted only bythe claims. It will be understood that the contents of patents, patentapplications, and literatures cited in the present specification shouldbe incorporated by reference to the present specification as if theircontents per se are specifically described in the present specification.The present application claims priority to Japanese Patent ApplicationNo. 2013-272084 (filed on Dec. 27, 2013) and it is understood that withregard to the contents of them, its contents should be incorporated byreference to the present specification as if its contents per se isspecifically described in the present specification.

INDUSTRIAL APPLICABILITY

Malignant tumor markers and malignant tumor control technologies areprovided and technologies applicable in industries (reagents, medicinemanufacture, and the like) involved in technologies related todiagnosis, therapy, and prophylaxis of malignant tumor are provided.

[Sequence Listing Free Text]

SEQ ID NO: 1: the anti-LSR antibody 9-7 sequence

SEQ ID NO: 2: the anti-LSR antibody 16-6 sequence

SEQ ID NO: 3: the anti-LSR antibody 26-2 sequence

SEQ ID NO: 4: the anti-LSR antibody 27-6 sequence

SEQ ID NO: 5: the anti-LSR antibody 1-25 sequence

SEQ ID NO: 6: the anti-LSR antibody 1-43 sequence

SEQ ID NO: 7: human LSR protein sequence (NP_991403.1)

SEQ ID NO: 8: human LSR nucleic acid sequence (NM_205834.3)

SEQ ID NO: 9: the core sequence (guide sequence) of LSR siRNA 1

SEQ ID NO: 10: the core sequence (guide sequence) of LSR siRNA 2

SEQ ID NO: 11: the antisense sequence of the core sequence (guidesequence) of LSR siRNA 1

SEQ ID NO: 12: the antisense sequence of the core sequence (guidesequence) of LSR siRNA 2

SEQ ID NO: 13: the sense full length sequence of LSR siRNA 1

SEQ ID NO: 14: the sense full length sequence of LSR siRNA 2

SEQ ID NO: 15: the antisense full length sequence of LSR siRNA 1

SEQ ID NO: 16: the antisense full length sequence of LSR siRNA 2

SEQ ID NO: 17: LSR forward primer sequence

SEQ ID NO: 18: LSR reverse primer sequence

SEQ ID NO: 19: β-actin forward primer sequence

SEQ ID NO: 20: β-actin reverse primer sequence

1. A method of treating or preventing a malignant tumor in a subject inneed thereof, comprising administering an effective amount of asuppressant of an lipolysis stimulated lipoprotein receptor (LSR) to thesubject, wherein the suppressant comprises an anti-LSR antibody or anantigen binding fragment or a functional equivalent thereof, wherein theanti-LSR antibody has positions 116-134 and/or 216-230 of SEQ ID NO: 7as an epitope.
 2. The method of claim 1, wherein the malignant tumor isan LSR positive malignant tumor.
 3. The method of claim 1, comprisingadministering the suppressant to the subject wherein the subject is apatient determined to have an episode of LSR positive malignant tumor.4. The method of claim 1, comprising administering the suppressant tothe subject wherein the subject is a patient among malignant tumorpatients whose malignant tumor has been determined to be LSR positivemalignant tumor.
 5. The method of claim 1, wherein the anti-LSR antibodyis an antibody having an ability to inhibit exacerbation due to a VLDL.6. The method of claim 1, wherein the anti-LSR antibody is an antibodyselected from a monoclonal antibody, polyclonal antibody, chimericantibody, humanized antibody, human antibody, multifunctional antibody,bispecific or oligospecific antibody, single chain antibody, scFV,diabody, sc(Fv)₂ (single chain (Fv)₂), and scFv-Fc.
 7. The method ofclaim 1, wherein the malignant tumor comprises ovarian cancer ormetastasized ovarian cancer.
 8. The method of claim 7, wherein theovarian cancer is recurrent ovarian cancer or early-stage ovariancancer.
 9. The method of claim 1, wherein the malignant tumor comprisesovarian cancer, pancreatic cancer, lung cancer, gastric cancer, or coloncancer.
 10. The method of claim 7, wherein the ovarian cancer is ovarianserous adenocarcinoma or ovarian clear cell adenocarcinoma.
 11. Themethod of claim 1, further comprising administering a cell-killingagent.
 12. The method of claim 1, wherein the anti-LSR antibody is oneor more antibodies selected from the group consisting of: (a) anantibody with heavy chain CDRs 1, 2, and 3 and light chain CDRs 1, 2,and 3 comprising amino acid sequences set forth in positions 31-35,50-66, 99-104, 153-165, 182-188 and 221-230 of SEQ ID NO: 1,respectively; (b) an antibody with heavy chain CDRs 1, 2, and 3 andlight chain CDRs 1, 2, and 3 comprising amino acid sequences set forthin positions 31-35, 50-66, 99-103, 152-165, 182-188 and 221-230 of SEQID NO: 2, respectively; (c) an antibody with heavy chain CDRs 1, 2, and3 and light chain CDRs 1, 2, and 3 comprising amino acid sequences setforth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and 221-229 ofSEQ ID NO: 3, respectively; (d) an antibody with heavy chain CDRs 1, 2,and 3 and light chain CDRs 1, 2, and 3 comprising amino acid sequencesset forth in positions 31-35, 50-66, 99-104, 153-165, 182-188 and221-229 of SEQ ID NO: 4, respectively; (e) an antibody with heavy chainCDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising amino acidsequences set forth in positions 31-35, 50-66, 99-104, 153-165, 182-188and 221-229 of SEQ ID NO: 5, respectively; (f) an antibody with heavychain CDRs 1, 2, and 3 and light chain CDRs 1, 2, and 3 comprising aminoacid sequences set forth in positions 31-35, 50-66, 99-104, 153-165,182-188 and 221-229 of SEQ ID NO: 6, respectively; and (g) a mutant ofthe antibody according to any one or more of (a)-(f), which is free of amutation in the CDRs but comprises one or several substitutions,additions, or deletions in a framework of the antibody in the mutant.